Download one-step and stepwise magnification of a bobbed lethal

Copyright 0 1986 by the Genetics Society of America
ONE-STEP AND STEPWISE MAGNIFICATION OF A
BOBBED LETHAL CHROMOSOME IN DROSOPHILA
MELANOGASTER
SHARYN A. ENDOW
AND
DONALD J. KOMMA
Department of Microbiology and Immunology, Duke University Medical Center,
Durham, North Carolina 27710
Manuscript received April 28, 1986
Revised copy accepted July 14, 1986
ABSTRACT
Bobbed lethal (bb‘) chromosomes carry too few ribosomal genes for homozygous
flies to be viable. Reversion of bb‘ chromosomes to bb or nearly bb+ occurs under
magnifying conditions at a low frequency in a single generation. These reversions occur too rapidly to be accounted for by single unequal sister chromatid
exchanges and seem unlikely to be due to multiple sister strand exchanges within
a given cell lineage. Analysis of several one-step revertants indicates that they
are X-Y recombinant chromosomes which probably arise from X-Y recombination
at bb. The addition o f ribosomal genes from the Y chromosome to the bb’
chromosome explains the more rapid reversion of the bb’ chromosome than is
permitted by single events of unequal sister chromatid exchange. Analysis of
stepwise bb’ magnified chromosomes, which were selected over a period of 4-9
magnifying generations, shows ribosomal gene patterns that are closely similar
to each other. Similarity in rDNA pattern among stepwise magnified products
of the same parental chromosome is consistent with reversion by a mechanism
of unequal sister strand exchange.
C
+
HROMOSOMES that carry severe deficiencies of the 18s 28s ribosomal
RNA genes are referred to as bobbed lethal (bb’) chromosomes (RITOSSA,
ATWOODand SPIEGELMAN
1966). bb’ chromosomes contain too few ribosomal
genes for bbl homozygotes to be viable. T h e number of ribosomal genes on a
bb’ chromosome must therefore increase more than twofold in order for the
chromosome to serve as the sole source of rDNA in a fly.
Progeny of flies which carry a low number of ribosomal genes show an
improvement in phenotype relative to their bobbed (bb) parents. This reversion of the bb phenotype has been termed “magnification” and is correlated
with an increase in rDNA in the bobbed mugnijied (bb”) chromosomes (RITOSSA
1968). Evidence from studies on ring chromosomes favors unequal sister strand
exchange as the major mechanism of ribosomal gene increase during magnification (TARTOF
1973b, 1974; ENDOW,KOMMA and ATWOOD1984; D. J.
KOMMAand K. C. ATWOOD,unpublished results). This evidence includes the
observations that magnification is inhibited in bb alleles in ring chromosomes
(TARTOF1973b, 1974; D. J. KOMMAand K. C. ATWOOD,unpublished results)
Genetics 1 1 4 511-523 October, 1986.
512
S. A. ENDOW AND D. J. KOMMA
and that ring chromosomes carrying bb alleles are lost under magnifying conditions compared with nonmagnifying conditions (ENDOW,KOMMA and ATWOOD 1984). Loss of ring chromosomes under magnifying conditions is correlated with an increase in abnormal ring structures, mostly dicentric chromosomes, that are attributed to products of sister chromatid exchange (ENDOW,
KOMMAand ATWOOD1984). Evidence consistent with an extracopy hypothesis
of rDNA magnification is the finding of extrachromosomal rDNA rings in
testes of magnifying males (GRAZIANI,
CAIZZIand GARGANO
1977), instability
and RITOSSA1970; BONof newly magnified bb (RITOSSA1968; HENDERSON
CINELLI et al. 1972; LOCKER1976) and the finding of amplified rDNA in GO
magnifying males (RITOSSAet al. 1971; LOCKERand MARRAKECHI1977; DE
CICCOand GLOVER1983). These observations are not unequivocal in their
support of an extracopy mechanism of magnification, however, since they can
also be explained within the context of an exchange hypothesis: The rDNA
rings could arise as products of intrachromatid excision (ENDOW,KOMMAand
ATWOOD1984), the instability could be due to chromosomes with which the
magnified allele is combined rather than to the magnifying event, and the
amplified rDNA might not contribute to the magnified phenotype. In contrast,
evidence from ring chromosome studies is not easily explained by the alternate
hypothesis: The failure of ring Xbb chromosomes to magnify cannot be easily
accounted for by the extracopy hypothesis since extracopies of rDNA should
be produced and integrated equally well into rod or ring X b b chromosomes,
whereas a lower recovery of bb" is predicted for ring Xbb chromosomes if
magnification occurs by an unequal sister strand exchange mechanism. This is
because sister chromatid exchange in ring chromosomes results in the formation of dicentric chromosomes and interlocked rings which are lost from the
cells that bear them (MCCLINTOCK
1938, 1941). The finding of the predicted
cytological structures and the observation that ring X b b chromosomes are lost
under magnifying conditions (ENDOW,KOMMA and ATWOOD1984) therefore
support unequal sister chromatid exchange as the major mechanism of magnification.
Some events that result in bb", however, are not easily explained by a sister
strand exchange mechanism of magnification: Under magnifying conditions bb'
chromosomes revert in a single generation with low but measureable frequencies
to bb or nearly bb+ (ATWOOD1969; RITOSSA1972). These onestep bobbed lethal magnified (bb'") chromosomes can be selected by their
ability to produce viable flies together with an X or Y chromosome that is
completely or almost completely rDNA-deficient. Magnification of bb' chromosomes to bb or nearly bb+ in a single generation cannot be due to single
unequal sister chromatid exchanges, since they can, at most, almost double the
starting number of ribosomal genes when pairing is in the most extreme register. The bb' chromosomes must more than double their ribosomal genes in
order to support viability as the sole source of rDNA for the fly. One-step
reversion of bb' chromosomes might occur by one or more of several mechanisms including X - Y recombination, as suggested by ATWOOD(1969), or by
some type of productive rDNA replication. A third possibility is that multiple
513
BOBBED LETHAL MAGNIFICATION
unequal sister strand exchanges occur in the same stem cell or premeiotic cell
lineage, producing bb' reversions in a single generation. We consider this unlikely to account for the one-step bb' reversions, however, since they are usually
recovered as single or low frequency (and therefore presumably meiotic) events
in individual bottles (ATWOOD1969; RITOSSA1973).
We have recovered and analyzed several one-step bb'" chromosomes. Our
results indicate that one-step bb'" chromosomes are X - Y recombinant chromosomes which probably arise as a result of X-Y recombination at bb. These
recombinational events can occur premeiotically or meiotically and are increased in frequency under magnifying conditions compared with nonmagnifying conditions. They represent a second, although low frequency, mechanism
to account for ribosomal gene increase during magnification and provide an
explanation for reversion of bb' chromosomes more rapidly than is permitted
by single events of unequal sister strand exchange.
During the course of this study we also analyzed several stepwise bb'" chromosomes. These stepwise bb' reversions were magnified in separate lineages
for 4-9 generations. Southern blot analysis of the final complement of ribosomal genes present in six stepwise bb'" chromosomes from two different lineages shows ribosomal gene patterns which are very similar to one another
and independent of lineage. This similarity in rDNA pattern among stepwise
magnified products of the same parental chromosome is consistent with an
unequal sister chromatid exchange mechanism of reversion.
MATERIALS AND METHODS
Drosophila stocks: Stocks of D.melanogaster carrying the y bb' and Ybby+chromosomes
were cloned in June, 1984 and October, 1982, respectively. T h e y bb' chromosome was
tested for lethality in homozygous females; n o y bb females were recovered. One-step
bb'" chromosomes were magnified in males and recovered in either females or males
according t o one of the following schemes. For recovery in females, y bb'/Ybb magnif y F g males were mated to + / Z n ( l ) s ~ ~ ~ys sc4
c ~ sc8
~ , car females. T h e Zn(1)sc4 2scBR,y sc4
sc car chromosome is an X-No chromosome; it is referred to as sc4sc8. Any y females
recovered among the progeny were expected t o be sc4scB/y bb'". These females were
tested for segregation of the bb and car markers by mating to sc4sc8/y+Y males, and the
y bb'" chromosomes were maintained as stocks of C(l)DX, y/y bb'"/y+Y. For recovery of
one-step bb"" chromosomes in males, y bb'/Ybby+ males were mated to C(l)RM, y 'U bb/
Ybb- females. T h e Ybb- chromosome carries a low number of ribosomal genes (TARTOF
1973a; SPEAR 1974; ENDOW 1982b) and has an g t i m o r p h i c effect with respect to bb
(MULLER1932). T h e C(1)RM chromosome is an XX chromosome. Any y male progeny
were expected to be y bbfm/Ybb-and were stocked as described above and tested further.
An experiment to test for one-step reversion of bb' chromosomes under nonmagnifying conditions was carried out by mating y bb'/Yy+ nonmagnifying males t o +/sc4sc8
tester females.
In order to recover stepwise bb" chromosomes, individual bb'/Ybby+ males were
mated each generation in single pairs to C(l)RM, y2 wa su(w")/Y2y + females in order t o
continue the magnification and also to sc4sc8/sc4sc8/BsYy+ tester females. y bb'/Y'bj+
males which showed an improvement in phenotype with respect to their fathers were
selected whenever possible in each successive generation to continue the magnification.
y bb'" chromosomes which were recovered in females with the sc4sc8 chromosome were
stocked and tested further. T h e lineage with respect to vial number of each of the
stepwise 66'" chromosomes that was recovered was recorded.
+
514
S. A. ENDOW AND D. J. KOMMA
T h e one-step bbIm chromosomes were tested for the presence of the Y chromosome
on the basis of fertility as X/O, X/y+YLand X / O ; T(Ys;4)/4males. T h e y+YL chromosome
(MULLER1948; NOVITSKI1952; SANDLER1954) was obtained from Bowling Green.
Males carrying y'YL but not Ys are sterile. T h e T(Ys;4) chromosome is a spontaneous
translocation recovered by D.J.K. It is Ks+,bb and homozygous lethal. Males carrying
T(Ys;4) but not YL are sterile.
Southern blot hybridizations: Crosses to produce sc4sc8/y bb" females were made by
mating y bb'"/y+Y males to +/sc4sc8 females in single pairs. Arpropriate crosses were
also made to produce y bb'/Ybby+Go magnifying males, sc4sc8/Y ?+ males, C(l)DX/Ybby+
females, y bb'/Y males, C( l)DX/Y females and +/y bb' females. Neuroblasts and imaginal
discs were dissected from y third instar female larvae, and DNA was prepared, as
described previously (ENDOWand GLOVER1979; ENDOW1982a), from pooled samples
of 3-15 sets of brains and discs and from 3-5 individuals from each vial. In one case,
DNA was prepared from adult brains in order to avoid sampling nondisjunctional
offspring. Pooled and individual samples of DNA were prepared from 2-3 single pair
matings for each genotype that was examined. rDNA blot patterns for pooled samples
were compared with each other and with patterns for individual samples for each of
the one-step and stepwise bb" chromosomes that were examined. T h e individual samples were used to confirm the rDNA blot patterns for each chromosome and to exclude
the possibility of tissue from contaminating genotype in the pooled samples. Ribosomal
gene repeats attributed to the y bb' chromosome were inferred from comparisons of
the rDNA blot patterns for bb'lyo' and C(I)DX, yf/YO', X°K/y bb' and X°K/XoK,and y
bb'/Y*?+ and C(I)DX, y wQ/Ylby+o r sc4sc8/Ybby+flies. T h e yo' and X°K chromosomes are
from o u r stocks of D. melanogaster Ore-R and OK- 1 (ENDOWand GLOVER1979; ENDOW
1980), respectively. T h e C(I)DX chromosome is an attached X chromosome that is
deficient for the nucleolus organizer re ion. T h e Ybby+rDNA pattern was obtained by
examining DNA from tissue of sc4sc8/Y y males o r C(l)DX, y wa/Ybby+females.
E+
DNA was digested with EcoRI o r BamHI restriction endonuclease, fractionated on
0.8% agarose gels, transferred to nitrocellulose and hybridized with ['*P]rDNA as described previously (ENDOW1980; ENDOW1982a). T h e rDNA probe was the 11.5-kb
intron- insert from pDmraa51 1 (DAWID,WELLAUER
and Long 1978; ENDOW1982a).
DNA quantitation: T h e concentrations of purified DNA in samples from pooled
tissue used for the Southern blot in Figure 2 were determined by ethidium bromide
fluorescence as described (MANIATIS,FRITSCHand SAMBROOK
1982), using bacteriophage lambda DNA samples of known concentration as standards. Briefly, 2-10 ng of
each DNA in a volume of 5 111 was mixed with 5 p1 of 2 pg/ml ethidium bromide in
distilled water; samples were photographed under shortwave ultraviolet illumination
onto Kodak Tri-X sheet film. Photographs were scanned using a Zeineh soft laser
scanning densitometer. Average maximum peak heights were determined for two scans
of each sample. A linear response of film density to ethidium bromide fluorescence was
found for DNA concentrations of 0.1-1.0 pg/ml. T h e amount of DNA in each sample
was determined relative to the bacteriophage lambda standards; the concentration of
DNA in each sample was calculated from the amount of DNA and the volume sampled.
Cytological preparations: Tissue squashes were prepared from third instar larval
neuroblasts from one-step and stepwise y bb'"/y+Y males, y bb'/BSY males, C(I)RM, y2 w0
su(w")/Y"y' females and B/Ybby+males by dispersing the tissue briefly in a drop of 45%
acetic acid on a siliconized coverslip and then squashing firmly onto a Denhardt-treated
slide (BRAHICand HAASE1978). Coverslips were flipped off after freezing in liquid
nitrogen, and slides were fixed overnight in absolute ethanol. Preparations were stained
Boehringer-Mannheim) and scanned under
with DAPI (4',6-diamidino-2-phenylindole;
fluorescence as described previously (ENDOW,KOMMAand ATWOOD1984).
RESULTS
Frequency of one-step reversion under nonmagnifying conditions: An experiment was carried o u t to determine the frequency of spontaneous one-step
515
BOBBED LETHAL MAGNIFICATION
TABLE 1
Frequency of one-step bb' reversion under nonmagnifying conditions
Offspring
I. Recovery in females
+?
Y?
dd
% bb"
14,756
0
20,236
<0.0068%
Progeny from cross of y bb'/Yy+ nonmagnifying males to +/In( I ) S C % ~ ~ , y sc4 sc8 cur IUI) females.
The one-step bb'" are recovered as y females; no y females were recovered in this experiment.
TABLE 2
Recovery of one-step bb'"' chromosomes under magnifying
conditions
Offspring
I. Recovery in males
11. Recovery in females
2
3666
+?
Y?
+d
7849
22
7716
Progeny from crosses of y bb'/Y"y+ magnifying males to C(I)RM, y U
bb/Ybb females (I) or +/Zn(I)sc'"~c~~,
y sc4 sc8 cur females (11). The onestep y bb" are recovered as y males (I) or y females (11). Twenty of the
22 y females in I1 arose in a single bottle.
reversion of bb' chromosomes. Nonmagnifying y bb'/Yy+ males were mated to
+/sc4sc8 tester females carrying the rDNA-deficient sc4sc8 chromosome. Offspring were examined for the occurrence of y females which were expected to
be y bb'"/sc4sc8. N o y females were recovered among 14,756 females that were
examined (Table l), indicating that the frequency of bbl reversion under nonmagnifying conditions is <0.0068%.
One-step bb" chromosomes: One-step y bb" chromosomes were magnified
for one generation in males and recovered in either males or females (Table
2). Selection of one-step y bb" chromosomes was by viability with an rDNAdeficient X or Y chromosome. Five independent one-step y bb'" chromosomes
were obtained from 11,539 flies that were screened. Two of the one-step y
bb'" chromosomes were recovered in males. One of these was sterile and could
not be examined further; the second was designated 10. Three one-step y bb"
chromosomes (35, 45 and 48) were recovered in females. Of these three, one
(48) was part of a cluster of 20 individuals from the same bottle. Three members of the cluster were saved and analyzed as described below. Results of this
analysis are consistent with the conclusion that the cluster of flies carrying onestep y bb" chromosomes arose from the same premeiotic event. T h e recovery
of 20 individuals in a single cluster indicates that the premeiotic event occurred
before the first mitotic division in sperm cell differentiation, i.e., during stem
cell division or differentiation. A total of four independent one-step y bb'"
chromosomes was analyzed.
516
S. A. ENDOW AND D. J. KOMMA
Cytological and molecular analysis of one-step bb Inr chromosomes:
Squashes of larval neuroblasts were prepared, stained with the fluorochrome
DAPI and scanned under fluorescence for metaphase figures. The y bb' chromosome is a typical rod-shaped X chromosome (Figure la) in which the short
arm is sometimes visible. The Ybby+chromosome consists of a long arm with
three brightly fluorescent regions and a short arm with two brightly fluorescent
regions (Figure lb). Similar observations have been made for other Y chromosomes stained with Hoechst 33258 or quinacrine (GATTI, PIMPINELLI
and
SANTINI1976; GATTI and PIMPINELLI1983). Three of the one-step y bb"
chromosomes, 10, 35 and 48, appeared as acrocentric chromosomes with a
uniformly staining long arm and a short arm consisting of three brightly fluorescent regions (Figure IC). T h e three chromosomes from the cluster of 20
y bb'" flies, 48-1, 48-3 and 48-4, were examined independently. Since they
showed the same cytological structure and genetic behavior, they are referred
to as 48. These one-step bb'" chromosomes, 10, ?5 and 48, were interpreted
to be X-Y recombinant chromosomes from their cytological appearance. Genetic tests confirmed that all three one-step y bb'" chromosomes carried YL
since they produced fertile males in combination with T(Ys;4), but not with
y'YL or in the absence of a Y chromosome. These cytological and genetic data
indicate that 10, 35 and 48 arose by an X-Y recombinational event that transferred Y L to the X chromosome.
The Southern blot hybridization rDNA patterns for the one-step y bb'" chromosomes were compared with the Ybby+,y bb' and y bb'/Ybby+ rDNA patterns
(Figure 2). This analysis indicated that part or all of the bb locus from the
Ybby+chromosome was present in the X-Y recombinant chromosomes. The
rDNA patterns for one-step y bb'" 10, 35 and 48 differed from one another;
however, the rDNA patterns for 48-1, 48-3 and 48-4 were identical to one
another. This observation, together with the cytology and genetic behavior of
48-1, 48-3 and 48-4, indicates that they most likely arose from the same reczmbinational event. Figure 2 (lanes a and b) shows the rDNA patterns for
XX-,/Ybby'
and y bb'/Ybby+flies. The rDNA bands in lane b attributed to the
y bbl chromosome are indicated with open circles. T h e y bb' chromosome was
also examined with the X°K and YOr chromosomes in order to confirm the
assignment of rDNA bands to the y bb' chromosome. It was necessary to derive
the y bb' rDNA pattern by subtraction of the rDNA bands from the bb or bb+
homologue, because the y bb' chromosome is lethal in homozygous form and
also with X-NO chromosomes, which would otherwise have been used for this
analysis. rDNA patterns for the one-step y bb'" chromosomes shown in Figure
2 are with an X-NO chromosome. The three one-step y bb'" chromosomes which
appeared cytologically to be X-Y recombinant chromosomes (10, 35 and 48)
contain rDNA bands characteristic of both the y bb' and the Ybby+chromosomes
(Figure 2, lanes c-e). One-step bb'" 48 (lane e) contains only a few ribosomal
genes that can be attributed to the y bb' chromosome, while I 0 and 35 contain
more. Repeats derived from the Ybby+chromosome are readily apparent in all
three one-step bb'" chromosomes. The DNAs in lanes b-e are present in approximately equal amounts.
FIGURE1.-Cytological structure of one-step y bb'" chromosomes compared with y bb' and p y +
chromosomes. a, The y bb' chromosome is indicated by the arrow. The brightly staining region is
the centromeric heterochromatin. b, The Y"y' chromosome is indicated by the arrow. Ys,which
is closer to the arrow, has two brightly fluorescent regions at its telomere; YL has three terminal
brightly fluorescent regions. The X chromosome shown here is bb+. c, One-step y bb'" chromosomes
10, 35 and 48 showed the structure indicated by the arrow, consisting of an acrocentric chromosome with a uniformly staining arm, a centromere and a short arm with three terminal brightly
fluorescent regions.
517
518
S. A. ENDOW AND D. J. KOMMA
7 kb
1.5
kb
h-4
FIGURES.--Southern blot rDNA patterns of one-step y bb" chromosomes. rDNA patterns for
the one-step y bb" chromosomes are shown together with patterns for the y bb' and u"y+ chromosomes. DNAs were digested with EcoRI. fractionated on a 0.8% agarose gel and transferred to
nitrocellulose. Hybridization was with a purified intron- rDNA repeat. The rDNA pattern for the
y bb' chromosome is derived by subtraction of the bands present in lane a from those in l z e b.
The positions corresponding to 11.5- and 17-kb repeats are indicated. DNAs are from a, XXw/
P y + ; b, y b b ' p y * ; c, one-step y bb" IO; d, one-step y bb" 3%e, one-step y bb" 48; and f. pDmr.
a5 1 1 (1 1.5-kb intron- rDNA repeat). DNAs in lanes b-e are from larval brains and imaginal discs
and are present in approximately equal amounts; DNA in lane a is from adult brains. The rDNA
bands attributed to the y bb' chromosome are indicated with open circles in lane b.
T h e fourth one-step y bb" chromosome that was examined, 45, appeared
cytologically to be a normal rod-shaped X chromosome. Its rDNA pattern did
not resemble that of the Ybby+ chromosome or the stepwise or one-step y bb"
chromosomes. T h e rDNA pattern for 45 could not be distinguished from that
for the X chromosome from our strain of D. melunogustet Ore-R when DNAs
from flies carrying the two chromosomes were compared on the same filter.
T h e most likely explanation for the recovery of the Xor chromosome as a onestep y bb" chromosome is that it was present in a nonvirgin tester female
which had mated with an Xo'/BsYy+ sibling male from the same cross from
which she arose. T h e Xor chromosome must have undergone a spontaneous
mutation to y in order for it to have been recovered in our screen, and the
female carrying the chromosome could not have been very fertile, because no
BS progeny were recovered from the bottle that produced 45.
Stepwise bb" chromosomes: Stepwise y bb" chromosomes were magnified
in males and recovered in females carrying an X chromosome deficient for bb.
BOBBED L E T N A L MAGNIFICATION
519
FIGURE3.-Southern blot rDNA patterns of stepwise y bbh chromosomes. rDNA patterns for
stepwise y bb" flies from two different lineages are shown. DNAs are grouped to show similarities
in rDNA patterns for flies from different lineages. DNAs were digested with EcoR1, fractionated
on a 0.8% agarose gel and hybridized with a purified intron- rDNA repeat as described in
MATERIALS AND METHODS. Lane f was from another gel. The lines indicate the bands which
correspond to those in lane f. DNAs are from stepwise y bb": a, 1-2-3-3-4; b. 6-2-3-1; c, 6-2-3-13-3-2-2-2; d, 6-2-3-1-2-1. e, 1-2-4-1-2 and f, 6-2-3-2.
Six lines of stepwise y bb" chromosome were established from single flies
arising from successive single-pair matings. These lines were designated 6-2-31, 6-2-3-2,
1-2-4-1-2,
1-2-3-3-4,
6-2-3-1-2-1
and 6-2-3-1-3-3-2-2-2,
with successive numbers indicating the vial for each generation and the total numbers
indicating the number of magnifying generations. T h e six lines represent two
lineages of y bb" chromosomes which were carried in different individuals
and 1-2-3-34),
three (6-2-3-1
and 6-2-3-2)
or four
after the first two (1-2-4-1-2
(6-2-3-1,
6-2-3-1-2-1
and 6-2-3-1-3-3-2-2-2)
generations. Stepwise y bb" chrowas recovered in an X-No/y bb" female after four magnifying
mosome 6-2-3-1
and 6-2-3-1-3,
were
generations; her half-sib y bb'/Ybby+ brothers, 6-2-3-1-2
fathered by the same male but apparently carried y bb' chromosomes of lower
rDNA redundancy than she did since they required two and five additional
generations, respectively, to produce x-NO/y bb" females.
Cytological and molecular analysis of stepwise bb" chromosomes: T h e six
stepwise y bb" chromosomes were examined cytologically, and all six were
found to be normal rod-shaped X chromosomes.
T h e rDNA patterns for the six stepwise y 66" chromosomes are shown in
Figure 3. T h e rDNA patterns for the six chromosomes are very similar, but
not identical, to one another. T h e small differences detectable among the
rDNA patterns are due primarily to different intensities of repeats in the 10to 12.5-kb range. There are also some rDNA bands of 4 0 k b that are present
in some patterns (a-c) but not others (d-9. No lineage-specific differences were
detected among the six stepwise bb" chromosomes examined in this study.
520
S. A. ENDOW AND
bb'
bby +
D. J. KOMMA
bb "
iy+
\
+
Y
Y
FIGURE4.-Origin of one-step bb'" chromosomes by X-Y recombination. One-step magnification
of bb' chromosomes can be accounted for by recombination between the X and Y chromosomes at
bb to produce X-Y recombinant chromosomes carrying YL.
Similarity in rDNA pattern between a starting bb chromosome and its magnified products has been reported previously (DE CICCOand GLOVER1983); in
this previous study, however, no differences in rDNA pattern among individual
flies were found after six magnifying generations.
DISCUSSION
O u r genetic, cytological and molecular analysis of three one-step bb" chromosomes indicates that they arose by an X-Y recombinational event in which
YL and bb, but not KS,were transferred to the X chromosome with the loss of
y+. Figure 4 shows a schematic diagram of an X-Y recombination that could
account for the chromosomes that we recovered. Because we detect different
amounts of X chromosome ribosomal genes in the rDNA patterns for the
recombinant chromosomes, we believe that the recombinations occurred at bb.
Our molecular analysis indicates that the recombinant rDNA patterns contain
repeats from both the starting X and Y chromosomes. The addition of ribosomal genes from the Y chromosome to the bbl chromosome explains the more
rapid reversion of the bb' chromosome than is expected for a single step of
unequal sister chromatid exchange. Our observation that reversion of a bbl
chromosome involves X-Y recombination indicates that bb reversion during
magnification may occur by a mechanism of homologous recombination in
addition to the previously described sister chromatid exchange (TARTOF
1973b, 1974; ENDOW,KOMMA and ATWOOD1984).
The experiments reported here demonstrate that both one-step and stepwise
bb" chromosomes can be recovered from flies of the same magnifying genotype. One-step bbim arises by X-Y recombination, probably at bb, whereas step-
BOBBED LETHAL MAGNIFICATION
52 1
wise bb'" most likely arises as a result of unequal sister chromatid exchange in
successive generations. The Ybb-chromosome, which is frequently used to establish magnifying conditions, is not required for magnification; other Ybb chromosomes, such as the Ybby+chromosome used in these experiments, also result
in magnification. The observation that other Ybb chromosomes will result in
magnification has been reported previously (ATWOOD1969; BONCINELLI
et al.
1972; HAWLEY
and TARTOF
1985; D. J. KOMMAand S. A. ENDOW,unpublished results; D. J. KOMMAand K. C. ATWOOD,unpublished results).
We observed a frequency of 0.035% for one-step bb' reversion (Table 2),
which is lower than previously reported values of 0.13-0.25% (ATWOOD1969;
RITOSSA1972, 1973). This might be due to our use of a more severe X and
Y combination than the ones used by others or to our ability to account for
clustering. We obtained 20 bb'" from a single bottle; cytological and molecular
analysis of three of the 20 suggests that the 20 bb" arose from a single premeiotic event. Clear clustering was not obtained in previous experiments; however, if some of the one-step bb'" flies obtained previously represented clusters
or portions of clusters, the frequencies of one-step events reported previously
would be overestimates. A control experiment that we carried out resulted in
a frequency of spontaneous bbl reversion of <0.0068%. The frequency of bb'
reversion under magnifying conditions is therefore increased at least fivefold
compared with nonmagnifying conditions. Thus, the recombinational events
that result in one-step bb' reversion are increased under magnifying conditions
compared with nonmagnifying conditions. Our data cannot be compared with
frequencies reported by others for X-Y recombination under magnifying or
nonmagnifying conditions, because only a subset of the total recombinants were
recovered, i.e., those that carried sufficient rDNA for viability with an rDNAdeficient X or Y chromosome.
Recovery of 20 bb" that probably correspond to a single event indicates that
the recombinational events resulting in one-step bb'" can occur early in spermiogenesis, i.e., in predefinitive stem cell divisions or during stem cell differentiation. Three other one-step bb'" chromosomes that were recovered probably arose during meiosis because they arose as single events in individual
bottles. The fifth chromosome recovered as a one-step bbim was a contaminating chromosome that was probably transmitted by a nonvirgin female. This
chromosome must have undergone a spontaneous mutation to yellow in order
to have been recovered in our screen.
T h e six stepwise bb" chromosomes that we examined showed very similar
but nonidentical rDNA patterns. Similarity in rDNA pattern among magnified
products is consistent with the generation of stepwise bb'" by sequential sister
chromatid exchange in successive generations. The small differences in rDNA
pattern that we observe for different stepwise bb" chromosomes can be attributed to different sites of exchange for events in each lineage. In addition, new
repeats could arise from unequal sister strand exchange at repetitive sites that
lie within a repeat, e.g., the internal repeats present in the nontranscribed
spacer regions of the Drosophila ribosomal genes (LONGand DAWID1979). In
these experiments we selected for sequential magnification products carrying
522
S. A. ENDOW AND D. J. KOMMA
an increased number of repeats. T h e prediction of crossover models in which
both increases and reductions in repeat number can occur is that the tandem
array will become homogeneous with respect to repeat type (SMITH 1973;
SZOSTAK
and Wu 1980). We are currently using molecular methods to examine
the effect of alternating cycles of magnification and reduction on a ribosomal
gene array.
These studies were supported by a grant from the National Institutes of Health (GM 31279)
to S.A.E. S.A.E. is a recipient of a United States Public Health Service Research Career Development Award (HD 00433). We thank K. C. ATWOODfor helpful discussions during the early
part of these studies.
LITERATURE CITED
ATWOOD,K. C., 1969 Some aspects of the bobbed problem in Drosophila. Genetics 61 (Suppl.):
319-327.
BONCINELLI,
E., F. GRAZIANI,
L. POLITO,C. MALVAand F. RITOSSA,1972 rDNA magnification
at the bobbed locus of the Y chromosome in Drosophila m.elanogaster. Cell Differ. 1: 133-142.
BRAHIC,M. and A. T. HAASE,1978 Detection of viral sequences of low reiteration frequency by
in situ hybridization. Proc. Natl. Acad. Sci. USA 75: 6125-6129.
DAWID,I. B., P. K. WELLAUER
and E. 0. LONG,1978 Ribosomal DNA in Drosophila melanogaster
I. Isolation and characterization of cloned fragments. J. Mol. Biol. 126 749-768.
DE
CICCO,D. V. and D. M. GLOVER,1983 Amplification of rDNA and type 1 sequences in
Drosophila males deficient in rDNA. Cell 32: 1217-1225.
ENDOW,S. A., 1980 On ribosomal gene compensation in Drosophila. Cell 22: 149-155.
ENDOW,S. A., 1982a Polytenization of the ribosomal genes on the X and Y chromosomes of
Drosophila melanogaster. Genetics 100 375-385.
ENDOW,S. A., 1982b Molecular characterization of ribosomal genes on the Ybb- chromosome of
Drosophila melanogaster. Genetics 102: 9 1-99.
ENDOW,S. A. and D. M. GLOVER,1979 Differential replication of ribosomal gene repeats in
polytene nuclei of Drosophila. Cell 17: 597-605.
ENDOW,S. A., D. J. KOMMAand K. C. ATWOOD,1984 Ring chromosomes and rDNA magnification in Drosophila. Genetics 108: 969-983.
GATTI, M. and S. PIMPINELLI,
1983 Cytological and genetic analysis of the Y chromosome of
Drosophila melanogaster I. Organization of the fertility factors. Chromosoma 88: 349-373.
GATTI,M.,S. PIMPINELLI
and G. SANTINI,
1976 Characterization of Drosophila heterochromatin
I. Staining and decondensation with Hoechst 33258 and quinacrine. Chromosoma 57: 351375.
GRAZIANI, F., R. CAIZZIand S. GARGANO,
1977 Circular ribosomal DNA during ribosomal magnification in Drosophila melanogaster. J. Mol. Biol. 112: 49-63.
HAWLEY,
R. S . and K. D. TARTOF,1985 A two-stage model for the control of rDNA magnification. Genetics 1 0 9 691-700.
HENDERSON,
A. and F. RITOSSA,1970 On the inheritance of rDNA of magnified bobbed loci in
D. melanogaster. Genetics 66: 463-473.
LOCKER,D., 1976 Instability at the bobbed locus following magnification in Drosophila melanogaster. Mol. Gen. Genet. 143: 261-268.
LOCKER,
D. and M. MARRAKECHI,
1977 Evidence for an excess of rDNA in the testis of Drosophila
melanogaster during rDNA magnification. Mol. Gen. Genet. 1 5 4 249-254.
BOEEED LETHAL MAGNIFICATION
523
LONG,E. 0. and I. B. DAWID,1979 Restriction analysis of spacers in ribosomal DNA of Drosophila melanogaster. Nucleic Acids Res. 7: 205-215.
MANIATIS,
T., E. F. FRITSCH, and J. SAMBROOK,
1982 Molecular Cloning: A Laboratory Manual.
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
MCCLINTOCK,
B., 1938 The production of homozygous deficient tissues with mutant characteristics by means of the aberrant mitotic behavior of ring-shaped chromosomes. Genetics 23:
315-376.
MCCLINTOCK,
B., 1941 The association of mutants with homozygous deficiencies in Zea mays.
Genetics 2 6 542-571.
MULLER,
H. J.. 1932 Further studies on the nature and causes of gene mutations. Proc. 6th Int.
Congr. Genet. 1: 213-255.
MULLER, H. J., 1948 The construction of several new types of Y chromosomes. Drosophila
Inform. Serv. 22: 73-74.
NOVITSKI,E., 1952 The genetic consequences of anaphase bridge formation in Drosophila. Genetics 37: 270-287.
RITOSSA,F. M., 1968 Unstable redundancy of genes for ribosomal RNA. Proc. Natl. Acad. Sci.
USA 6 0 509-516.
R"A,
F. M., 1972 Procedure for magnification of lethal deletions of genes for ribosomal
RNA. Nature (New Biol.) 2 4 0 109-1 1 1 .
RITOSSA,F. M., 1973 Crossing over between X and Y chromosomes during ribosomal DNA
magnification in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 7 0 1950-1954.
RITOSSA,
F. M., K. C. ATWOODand S. SPIEGELMAN,
1966 A molecular explanation of the bobbed
mutants of Drosophila as partial deficiencies of "ribosomal" DNA. Genetics 54: 819-834.
RITOSSA,F. M., C. MALVA,E. BONCINELLI,
F. GRAZIANI
and L. POLITO,1971 The first steps of
magnification of DNA complementary to ribosomal RNA in Drosophila melanogaster. Proc.
Nat. Acad. Sci. USA 68: 1580-1584.
SANDLER,
L., 1954 Heterochromatic exchange between reversed acrocentric compound X chromosomes and FR2. Drosophila Inform. Serv. 28: 153-154.
SMITH,G. P., 1973 Unequal crossover and the evolution of multigene families. Cold Spring
Harbor Symp. Quant. Biol. 3 8 507-513.
SPEAR,B. B., 1974 The genes for ribosomal RNA in diploid and polytene chromosomes of
Drosophila melanogaster. Chromosoma 4 8 159- 179.
SZOSTAK,
J. W. and R. Wu, 1980 Unequal crossing over in the ribosomal DNA of Saccharomyces
cereuisiae. Nature 284: 426-430.
TARTOF,K. D., 1973a Regulation of ribosomal RNA gene multiplicity in Drosophila melanogaster.
Genetics 73: 57-71.
TARTOF,K. D., 1973b Unequal mitotic sister chromatid exchange and disproportionate replication as mechanisms regulating ribosomal RNA gene redundancy. Cold Spring Harbor Symp.
Quant. Biol. 3 8 491-500.
TARTOF,K. D., 1974 Unequal mitotic sister chromatid exchange as the mechanism of ribosomal
RNA gene magnification. Proc. Natl. Acad. Sci. USA 71: 1272-1276.
Communicating editor: A. SPRADLINC