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Date

0-7639

May 83

                                                                                                                               

1293 00896 3765

This is to certify that the

thesis entitled

Test for Rex and Su(Rex) in
natural populations of D. melanogaster

presented by
Mekki Boussaha

has been accepted towards fulfillment
of the requirements for

MaSter l 5 degree in ZOO] Ogy

 

 

/ Major éiessor

1991

MS U is an Affirmative Action/Equal Opportunity Institution

= “mum. *,-' -_~- -

 

 

LIBRARY
Michigan State
University

\_ J

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU Is An Affirmative Action/Equal Opportunity Institution
cicithpmS—pn

 

TEST FOR REX AND SuiREX)

IN NATURAL POPULATIONS OF Q. MELANOGASTER

BY

Mekki Boussaha

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Zoology

1991

‘ I
5/ 7/

f :54 -

ABSTRACT

TEST FOR REX AND SulREX)
IN NATURAL POPULATIONS 0F 2; EELANOQASTER.

BY

Mekki Boussaha

3g; (Bibosomal exchange) is a genetic element of
Drosophila melanogaster that induces mitotic recombination in
the ribosomal RNA genes (rDNA, nucleolus organizer, bb locus).
In. the course: of this study some features of Igex; were
characterized.
(1) Crosses and progeny counts were used to test some sixteen
wild-caught X chromosomes from a world-wide sample of
populations for Be; activity, as well as for suppressors of
Rex (Su(Rex)). The results show that.3ex;was not detectable in
natural populations whereas Su(Rex) was present in all
samples. These observations leave open the question of whether
3g; is present in wildtype Drosophila since the presence of
Su(Rex) precludes knowing whether Beg was present.
(2) Rex-induced exchange can be used to construct rDNA maps
quickly, but we need to measure rDNA copy number reliably for
fairly large numbers of samples. Tecniques for rapidly
determining rDNA copy number were, therefore, examined. A
single-insect squash-blot technique while promising, shows
too 'much 'variation. Quantitative dot—blots, however, are

reliable.

 

To my parents

for their support and love

to Leonard G. Robbins

for his support psychologically, academically and financially

 

ACKNOWLEDGMENTS

I am grateful for the superb technical assistance,
precious advice and, encouragment of Dr. Leonard G. Robbins.
I also thank Dr. Thomas Friedman and Dr. Larry Snyder for
their critical reading of the manuscript. I am grateful to Dr.
Scott Williams. His promptness in sending requested stocks
greatly facilitated the research presented here. Special
thanks are also due Susan Lootens, Robin Steinman, Rob Morell,
Lorri Wallrath, Peter Crawley and Mark Thompson for their help
and for being’ wonderful officemates. I also thank Ellen
Swanson for her patient assistance.

Finally, I have dedicated this work to my family, and

here add my special thanks to all my friends back home.

iv

TABLE OF CONTENTS

 

LIST OF FIGURES. ...... .. ................................... Vii
LIST OF TABLES.......................... ........... .......Viii
INTRODUCTION............... .......... .. ..................... .1
Areas of investigation ..... ...... ........... ............3
CHAPTER 1 . LITERATURE REVIEW. . . ........... . ............... . . . 4
The rDNA of Q. mlanogaster. . . . ......................... 4
Concerted evolution..... ................................ 6
Lag-induced events. ......... . ........................... 7

The responding site of M ........................ 12

The nature of 3% ...... . . ......................... 12

Mapping of M and Su (Rex) ........................ 12

Rex induces spiral and hairpin exchanges .......... 13

Deletions generated by M ........................ 13

CHAPTER 2 . MATERIALS AND METHODS
Fly stocks and rearing methods .............. . . . . . . . . . . . 16
Wildtype stocks ................................. . . 16

Crossing schemes

Tests for Rex activity in natural populations ..... 18
Tests for Su(Rex) in natural populations .......... 18

CHAPTER 3. RESULTS AND DISCUSSION

Results
Tests for Bi); activity in natural populations ..... 24
Tests for Su(Rex) in natural populations .......... 24
Discussion ............................................. 24

V

APPENDIX. MEASUREMENT OF rDNA COPY NUMBER. . . . . .............. 29

MATERIALS AND METHODS

Separation of parental NO's ....................... 31
Separation of recombinant NO's ................. . . .31
DNA dot blot ...................................... 33
Single-insect squash blot ......................... 34
Radiolabelling of DNA probes ...................... 35
Probe-labelling and hybridization ......... . ...... .35
Hybridization kinetics ............................ 36
Calculation of 10x excess of probe ................ 37
Counting and calculations ......................... 38

Counting ..................................... 38

Calculations ................................. 39

PRELIMINARY RESULTS
Squash blot ....................................... 40
Improving the efficiency of binding and
keeping the dots small .................... 4 0

Different ways of fixing the DNA onto

filters .................................. 43

Linearity .................................... 43

DNA dot blot ...................................... 46

Different ways of denaturing the DNA ......... 46

The time period of pre-hybridization ......... 49

The time period of hybridization ............. 49

CONCLUSION ............................................. 54
BIBLIOGRAPHY ................................................ 55

vi

Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure

Figure

LIST OF FIGURES

Schematic representation of the X chromosome ....... 2
Map ofasingle DNA repeat unit........... ...... ...5

Detection of Rex activity in natural populations. . .9

 

Synthesis of 12:12 ............................... 11
Two types of R_ex-induced exchanges. ............... 15
Test for flex activity in natural populations ...... 20
Test for Su(Rex) in natural populations ........... 23
Separation of parental NO's ....................... 30
Separation of recombinant NO's .................... 32

Figure 10. Hybridization of genomic DNA from squashed

YSX‘YL/Q males and C(11RM/Q females ........... 41

Figure 11. Hybridization of genomic DNA from squashed

wfgiflies probed with rDNA sequences ......... 42

Figure 12 . test for linearity ............................... 45

Figure 13. Denaturation procedure and pre-hybridization

time. ................................ . ........ 48

Figure 14. Ore-R DNA blots hybridized with rDNA probes for

one, two, three or four hours ................. 50

Figure 15. Ore-R DNA dot blots hybridized with U0 probe

for four or 18 hours .......................... 53

Vii

LIST OF TABLES

 

Table 1. Origin of wildtype Drosophila. ..................... 17
Table 2. Test for & activity in natural populations ....... 25
Table 3. Test for Su(Rex) in natural populations ............ 27
Table 4 . Test for fertility ................................. 28

viii

INTRODUCTION

Genetic and physical maps of chromosomes have provided a
framework for further studies in ‘molecular biology. The
heterochromatin, however, has been refractory to conventional
mapping techniques. Even though, for example, 40% of the
length of the Drosophila X chromosome and all of the X
chromosome are heterochromatic (Hilliker et al., 1980), the
large-scale organization of heterochromatin remains mysterious
for a variety of reasons: (1) there are very few phenotypic
markers; (2) the recombination frequency is extremely low
(approximately 0.02% for the z heterochromatin), and (3)
repeated gene families within the heterochromatin are too
large to be cloned using conventional vectors.

Rex-induced recombination in the ribosamal RNA genes
(rDNA) of Drosophila melanogaster is at least two-orders of
magnitude more frequent than spontaneous events. Moreover, the
rDNA is located in the X heterochromatin and occupies
approximately 40% of its physical length (Fig. 1) (Hilliker et
al., 1980). The rDNA is, therefore, a model gene for
establishing a methodology for mapping repeated genes in the

heterochromatin (Williams et al., 1990).

 

SUN)
y sc w sp/ cv lz v I BBX car
\Ilit’ 1 I 1 \11 1 1 .
II II I l l Ir 1 1

5 map units

 

Figure 1 - Schematic representation of the X chromosome

The X chromosome is 66 map units long, and the map positions
of many of the phenotypic markers are shown. The basal
heterochromatin (black boxes) is approximately 40% of the
physical length (Hilliker gt g;., 1980; Schalet & lefevre,

1976; Williams gt al., 1989). The NO occupies about one—third

of the basal heterochromatin and is shown as an open box.

 

AREAS OF INVESTIGATION

There are several questions about Rex, and, more

 

importantly, about the rDNA that 3g; can be used to answer.

Two main questions have been addressed. The first forms the
body of this thesis, the second is discussed in the appendix:
(1)- Is 3g; present in natural populations? Egg—induced rDNA
recombination is at least two orders of magnitude more
frequent than spontaneous events. If 3g; is present in natural

populations, Rex-induced exchange would be a major factor in

 

the concerted evolution of this gene family.

(2)- Rgt-induced exchange events can be used to map the
structure of the rDNA array. Improving the resolution of our
rDNA maps, as well as being able to construct these maps
quickly using Egg-induced exchange, depends on being able to
determine rDNA copy number for fairly large numbers of
samples. An attempt was made to devise a reliable and rapid

technique for measuring rDNA copy number.

CHAPTER 1

LITERATURE REVIEW

I) - The rDNA of Drosophila melanogaster

In wild—type D. melanogaster there are two clusters of
repeated rRNA genes (rDNA), each with approximately 250 copies
(Long and Dawid, 1980; Tartof, 1973a). One of these is located
in the proximal heterochromatin of the X chromosome and the
other is located on the short arm of the X chromosome. These
two rDNA arrays correspond genetically to the bobbed (pp)
loci. A single repeat unit contains coding regions for the 28,
5.88, 188, and 288 rRNA subunits (Fig. 2) (Tautz gt gt.,
1988). The series of alleles known as bobbed represent the
range of subnormal numbers of these genes (Ritossa, Atwood and
Spiegelman, 1966). Bobbed flies show delayed development and
have a phenotype that includes small thoracic bristles,
abdominal etching and, in severe cases, malformed genitalia
(Lindsley and Zimm, 1985). The most extreme alleles are
lethal.

Some of the rDNA repeats are interrupted in the 28S
coding region by one of two types of nonhomologous insertion
sequences. In two common laboratory wild-type stocks (Oregon—R
and Canton-S), type 1 (T1) insertions are restricted to the X
chromosome where they interrupt about 50% of the rDNA repeats.

The DNA sequences at rDNA/T1 junctions and the location of

Han-cubed
spacer 188 5.88 23 288 lnter~genlc spacer

 
 
 
 

  
 

Hincli Hlndlll Haelll

    

Haelll

neerflon sequences
In I I Type I
BamHl

-IIIIII?Il-Typell

T T

EcoRl

Figure 2 — Map of a single rDNA repeat unit.

The NO contains approximately 250 rDNA repeats or cistrons
(Tartof,1973a). A single cistron is shown schematically, with
the transcribed sequences in boxes. There are coding regions
for four species of rDNA, and two transcribed spacers shown as
open boxes, and a non-transcribed inter—genie spacer (IGS)
(Tautz gt _;., 1988). Some cistrons in each NO are interrupted
in the 28S coding region by insertion sequences which are
classified as either type I or type II depending on sequence.
Type I insertions appear to be unique to the z chromosome,
while type II insertions are found both in )( and 1 NO's

(Wellauer gt

1., 1978). Each insert class has a specific
integration site in the 288 coding region, and the two sites
are separated by fewer than 100 bp (Roiha gt g;., 1981). The
dashed lines within the insertion sequences and within the IGS

indicate the length of these regions.

6
sequences homologous to‘Tl insertions away fromrthe nucleolus
organizer suggest that these elements may be transposable
(Kidd and Glover, 1981; Peacock gt gt., 1981; Roiha gt g;.,
1981). Type 2 (T2) insertions interrupt about 15% of the rDNA
repeats on both the z and X chromosomes. These insertions are
not homologous to T1 insertions and have a slightly different
insertion point in the 288 rRNA coding region (Figure 2)
(Roiha and Glover, 1981; Long and Dawid, 1980). Repeats
containing T1 and T2 insertions do not appear to contribute
significantly to the production of mature rRNA (Long and
Dawid, 1979; Long gt g;., 1981)

Another source of heterogeneity within.rDNA arrays is the
non—transcribed or intergenic spacer (IGS) which.separates the
repeat units and is highly variable in length, ranging from
4kb to 20kb (Coen gt _a__l_., 1982; Indik and Tartof, 1980;
Terracol, 1986). The 5' portion of the IGS contains a series
of 340bp sequences bounded by Egg; sites (Williams gt gt.,
1987); whereas the central portion of the IGS contains a
variable number of 240bp sequences bounded by Alp; sites (Coen
._t _;., 1982; Simeone gt gt., 1985)

II) - Concerted evolution

Evolution of repetitive gene families presents a serious
problem. Independent mutations would be expected to yield
divergence of members of a repeated array. However,

homogeneity is most common. For Drosophila rDNA, although

7
there are diagnostic differences between the z and X
chromosomes, they too are quite similar. Moreover, the
transcribed segments, within a chromosome, are nearly
identical. Through genetic interactions among its members, the

gene family may evolve together in concerted fashion, tg. as
a unit (Arnheim, 1983). Mechanisms of molecular exchange,
including unequal crossing over and gene conversion, are
thought to account for concerted evolution (Dover, 1982;
Arnheim, 1983; Smith, 1973; Arnheim gt g;., 1980; Tartof,
1988) because they are capable of rapidily homogenizing
selectively neutral mutations. In the case of the Drosophila
rDNA, however, analysis of spontaneous rDNA exchange indicates
that reciprocal recombination and gene conversion are not
sufficient to explain the observed patterns of homogeneity and
difference found in natural populations (Williams gt gt.,
1989).

Because Egg-induced rDNA recombination is at least two
orders of magnitude more frequent than spontaneous events, if

Rex is present in natural populations, Rex-induced exchange

 

would be a major factor in the concerted evolution of this

gene family.

III) - Rex-INDUCED EVENTS
Rex is a genetic element of D. melanogaster that induces
mitotic exchange in the ribosomal RNA (Robbins, 1981). Rex was

not discovered as a new mutant. Rather, it was detected in the

 

Figure 3 — Detection of fig; activity in natural populations

A: The attached-XX target chromosome is schematically shown
undergoing a Egg-induced exchange event. This exchange leads
to loss of z euchromatin and detachment of a complete X
chromosome from the attached—XX chromosome. Heterochromatic
regions are indicated by thick lines and the pp loci are
indicated by open boxes.

5: The punnet square for a typical Rgx mating is shown. The
Egg—induced exchanges take place in fig; female zygotes,
yielding XX sons or gynandromorphs. These are readily

distinguished from regular sons because of the y: marker.

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10

X chromosomes of a Df(1)wrJl stock. This chromosome was first
isolated as an unequal crossover in the y region. Aside from
the exchange that generated the deficiency, the provenance of
this chromosome is unrecorded. We, therefore, do not know the
origin or molecular basis of Egg, but we can determine its
properties and from that make inferences about both its origin
and nature.

Egg is a maternal effect dominant locus (hence, probably
encodes a product). It was detected because Rgx mothers
induced mitotic exchanges in their offspring between two
ribosomal DNA arrays in an attached-XX chromosome. The result
is the production of free X chromosomes (Fig. 3) (Robbins,
1981). The exchange event is mitotic and takes place in the
early zygote, either before S of the first cell cycle changing
5&1 daughters into 3X sons, or after 8 of the first cell
division or at the two cell stage producing gynandromorphs
(Fig. 3) (Robbins, 1981).

The attached-31 chromosome in which the Egg-induced
detachment was first detected is l2flflj(Lindsley and Novitski,
1959). Fig. 4 shows the structure and origin of this
chromosome. The short arm of the 1 (X5 is attached to the
distal z euchromatin, and the long arm (it), marked with a
small translocation of the z euchromatin carrying' yj, is

attached to the centromere.

 

 

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12
1) - The responding site of Rex - Swanson (1984) demonstrated
that 3g; causes exchange between any two separated blocks of
rDNA on a single chromosome. She tested various chromosomes
with two rDNA.blocks for ability'tO'undergo»detachment events.
Even half a block of rDNA at each end of the chromosome can be
a target. She has also shown that simple duplications of
heterochromatin do not constitute a _Re_x target; In(1)wm4 which
has type 1 insertion sequences at both ends of the chromosome
(Hilliker and Appels, 1982) is not a target. These
observations leave open the question of whether an exactly
duplicated non-rDNA block of heterochromatin at both ends of

a chromosome can act as a Rex target.

2) - The nature of Rex - Dosage studies indicate that, using
the classification scheme proposed by Muller (1932), 3g; is a
neomorph.or extreme hypermorph (Rasooly and.Robbins, 1991). It
therefore produces a novel function, or a normal product that

is abnormally expressed.

3) - Mappinqt of Rex and Su(Rex) — Egg maps to the
heterochromatin of the X chromosome and its target is
specifically the rDNA. Egg and a suppressor of Rex (Su(Rex))
not only map to the nucleolus organizer, but also map as
repeated elements at discrete locations in the rDNA array

(Rasooly, 1989; Rasooly and Robbins, 1991).

13
4) - Rex induces spiral and hairpin exchanges — Rgx generates
two kinds of exchanges, depending on whether the target
chromosome pairs in a "spiral" or a "hairpin" configuration
(Robbins and Swanson, 1988). When the target chromosome pairs
as a spiral, the product is deleted for all of the material
originally in between the two nucleolus organizers. If the
deletion removes most of the X chromosome euchromatin, these
products can, with the appropriate markers, be readily
detected as phenotypically distinctive males or
gynandromorphs. When the nucleolus organizers pair in the
opposite orientation, the hairpin configuration, the exchange
inverts the material between the nucleolus organizers rather
than deleting it. A typical mating, the Egg-induced events,

and their products are shown in Fig. 5.

5) — Deletions generated by Rex - Egg generates deletions at
the exchange sites in the rDNA that can be identified both
molecularly and genetically. These observations suggest that
3g; may be an active version of one of the insertion sequences
found in the rDNA. It could encode a site-specific
endonuclease activity causing recombinogenic breaks in the
target DNA. Resolution of these breaks would then lead to the
exchanges we see, as well as to deletions at the exchange

site.

14

Figure 5 - Two types of Egg-induced exchange

The target shown is an X chromosome duplicated for the NO and
surrounding heterochromatin. Maternal Rgx activity induces
both types of exchange in the target chromosome in a single
cross (Robbins & Swanson, 1988). The spiral exchange deletes
the intervening X euchromatin, changing z/z daughters into

sterile z/fragment males patroclinous for only y: or

gynandromorphs with y: male tissue. The hairpin exchange

simply inverts the material between the two NO's.

15

 

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CHAPTER 2

MATERIALS AND METHODS

I) - FLY STOCKS AND REARING METHODS

All flies for stocks. and. crosses ‘were .reared. on .a
standard Drosophila medium of cornmeal, molasses and brewer's
yeast at ZFTL Mass matings were done in polyethelene bottles,
15 males with 15 females. Matings for counting offspring and
testing for fertility were done in glass shell vials, one
female with three males and one male with three females,
respectively. Unless otherwise noted, all phenotypic markers
and standard chromosomes are described in Lindsley and Grell

(1968) and Lindsley and Zimm (1985, 1987).

1) Wildtype stocks - Seventeen Drosophila stocks carrying
wildtype X chromosomes were obtained from Scott Williams on
July 9, 1990 (Table 1). These stocks come from a total of nine
isofemale lines. The original isofemale lines were collected
from Argentina (Arg4; Arg6), Australia (Aust.Bl-lo; Aust.Bl-
_1); Central African Republic (Caf—15); Taiwan.(Taiwan-20) and
Vietnam (Viet. 13—I, Viet. 15-I). From each of these original
lines, males were crossed individually to virgin females with
compound X chromosomes and sons and daughters were mated

(Williams, 5., personal communication). Thus, all males in the

stocks we received are isogenic for their 3 and X chromosomes.

16

17

 

ISOFEMALE LINES

OBSERVATIONS

 

ARGETINA

Arg—4 I
Arg-4 II
Arg—4 IV
Arg-6 I

Arg-6 II

Stock died

 

AUSTRALIA

Aust BL-lO I

Aust BL-lO II

Aust BL-l7 IV

Aust BL-17 V

 

CENTRAL

AFRICAN

REPUBLIC

CAL-15 I

CAL-15 II

 

TAIWAN

Taiwan-20 I

Taiwan—20 II

 

 

VIETNAM

 

Viet 13‘1 I
Viet 13-1 II
Viet 15-1 I

Viet 15-1 II

 

 

Table 1 - Origin of wildtype Drosophila.

 

18
The Roman numeral for each stock (e.g., Taiwan go-II)
signifies that it was from the second male. All the isofemale

lines have more than one representative.

II) - CROSSING SCHEMES

1) Tests for Rex activity in natural populations - The mating
scheme used to test for Egg-activity in natural populations is
shown in Fig. 6. Homozygous y w _s_p_l_ virgin females are crossed
to wiltype males and the F1 1/1 E gp; female offspring are
crossed to males carrying the target chromosome, 'Xfltgfi,
In(1)EN, y y t §;y*/Q. Regular males are z/Q and, therefore,
sterile. Non-disjunctional males are \_I t I_3_ and are
distinctive. y: E males are generated by Egg-induced mitotic
events. A crossover between y and y, however, also produces y_+
w males. These are 3/9 and sterile while the yf y products of
Re_x activity would be z/yfl and fertile. All y: w males
recovered are, therefore, tested for fertility. Recovery of

fertile males would reveal the presence of Rex in the

corresponding wildtype stock.

2) Test for Su(Rex) in natural populations - Fig. 7 shows the
mating scheme used to test for Su(Rex) in sixteen of the wild-
caught X chromosomes. 3g; is kept in stocks as BEE/X males x
C(1)DX/1 females. EM/gn Ex females were crossed to y _w _sp; g

were then crossed to wildtype males and the wildtype female

 

19

Figure 6 - Test for Rex activity in natural populations.
Homozygous y y 591 virgin females were crossed to wildtype
males and the F1 +/y E 591 female offspring were crossed to

YSX'YL, IN(1)EN, y y t B' yj/Q males.

 

20

 

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21
offspring were then crossed to _YS_X'Y_L, In(1)EN, y y t §.y:/Q
males. y: E males, that are either yi/Q crossovers between y
and g, or Egg-induced z/ij, were scored and then tested for
fertility. The y_+ tr males produced by Rex-induced mitotic
events would be fertile. If Su(Rex) was present in a

particular stock, no fertile y: males would be detected.

 

22

Figure 7 - test for Su(Rex) in natural populations.

wildtype males and the wildtype female offspring were then

crossed to YDCWfl IN(1)EN, y y t g yj/Q males.

23

 

 

 

 

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CHAPTER 3

RESULTS AND DISCUSSION

I - RESULTS

1) Tests for Rex activity in natural populations - Table 2
shows the total progeny counted for each isofemale line of
Drosophila melanogaster. Of a total of 358 y: E male progeny
(Table 4), however, all were sterile. These observations

suggest that Rex was not detectable or was not present in

natural populations.

2) Tests for Su(Rex) in wildtype Drosophila - Total progeny
counted for each isofemale line are shown in Table 3. Of a
total of 401 y: w male offspring scored (Table 4), none has
proven to be fertile. These observations imply that Su(Rex) is

wide—spread in natural populations.

II - DISCUSSION

Two possibilities can be considered to explain the
absence of fig; activity in the X chromosomes derived from
these populations:
(1) - 3g; is not present in natural populations. Rather, it is
limited to some laboratory stocks, hence, 3g; can not be
considered as a major factor in concerted evolution of the

rDNA gene family.

24

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26

(2) - Egg might be present in wildtype Drosophila, but it is
heavily suppressed by Su(Rex). The presence of Su(Rex) in
natural population does not, however, necessarily imply the
presence of Egg since a suppressor may have other functions as
well as suppressing Egg.

Su(Rex) was, indeed, present in all of the samples. These
observations leave open the question of whether Egg is present
in wildtype Drosophila since the presence of the suppressor
precludes knowing whether Egg is there as well. One way to
resolve this question would be to define the function(s) of
Su(Rex). If Su(Rex) has only one function, to suppress Egg
activity, then the presence of the suppressor would imply that
Egg is also wide-spread in natural populations. In that case,
Egg could be involved in the concerted evolution of the rDNA
gene family. If Su(Rex), however, has more than one function,
then we would still not know whether Egg is present in natural

populations of Drosophila.

27

 

+

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

$536.. E» H m mm» Fa. an? M m w HI m E m». H m...
fivunm mmEuHmm KNHmm Kappa wnamwmm Raven Keven Haven
fiHQIa HHH thu quo Hooq H A NN 0 HQ
VHQlaa H4 uNON HmQH NONH N m NN o Hm
vHle H NmHm Hmmm quH u A Nm O HO
>Hfllm HH wmwm HmQH wam w m NH 0 Ho
56¢" wblHo H woeN Hmuw NOOA N N H@ o Hm
56m" wfiIHu HH quq Hme NuwH H A NN 0 PW
Fawn wHIHu H4 upmq Noqm NNNu N m N@ o Ho
Vfimn wfitwu < wowH Hmww NHqu u m Nm 0 Ne
ODHIHW H uwoN Hmmm quq N 3 Nm 0 Ho
ONHIHM HH Nme Hflom NNqo u m Na 0 NN
HflwtflblNo H wNmm Hume NHmo o H Nu 6 Ho
Hewtwuimc HH ammo Hmmm Nmmm H m we 0 pm
<Mmflin|H H NmmH Hqu Hmmm N M NH o NN
dwmnIHmIH HH mmmo Home .mmmm m a mm 6 mo
dwwanMIH H NON» Hqu Hmmm w m Nu o NH
dwmfllHMIH HH uqu Hme NHmm H w Nm 0 No

 

 

 

 

 

 

 

 

 

HASHe u i ammfl Hon mcnwmxv H: smncnmw popcwwflwosm on U. BmHmdoomman.

 

28

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

es—x _wa
Total Fertile Total y: g Fertile
y: 31 y: w y: u
Arg-4 III 21 0 22 0
Arg—4 IV 27 0 22 0
Arg—6 I 17 0 28 O
Arg-6 II 21 o 21 0
Aust BL-lO I 21 O 19 0
Aust BL-l7 II 26 0 22 0
Aust BL-l7 IV 23 O 29 0
Aust BL-17 V 23 O 25 0
CAL-15 I 24 0 26 O
CAL-15 II 16 0 26 O
Taiwan-20 I 21 O 23 O
Taiwan-20 II 23 O 30 0
Viet-13-l I 24 0 27 O
Viet-l3-l II 17 0 33 O
Viet-lS-l I 33 0 23 O
Viet-lS-l II 21 O 25 0
TOTAL 358 O 401 O

 

 

 

 

 

 

 

Table 4 — Testing y: g males for fertility.
y: g males were crossed to C(1)RM, y y pp females. y males and

y females are expected from this cross if the male is a y

F+

g/y+Y detachment product.

APPENDIX

MEASUREMENT OF rDNA COPY NUMBER

Improving the resolution of rDNA maps, as well as being
able to construct these maps quickly using Egg-induced
exchange, depends on being able to determine rDNA copy number
reliably for fairly large numbers of samples. I have,
therefore, devoted some time to technology development. At
first, a quantitative single-insect squash-blot technique (
Betty gt gt., 1988; Tchen gt _t., 1985) appeared promising,
but I have found it to be too variable. I have, therefore,
turned to quantitative dot blots. The results seem to be
reliable.

Once the parameters of the technique have been
established, twenty-two pairs of chromosomes will be tested;
a parental pair and eleven pairs of recombinants from one
series of hairpin exchanges and a parental pair and nine
recombinant pairs from a second series. The first series is

described here.

29

 

MATERIALS AND METHODS

I) Separation of parental NO's

 

Two chromosomes will be tested: In(1)wm4 (ESQ and
In(l)w‘“5"’ (fl) . As shown in Fig. 8, the breakpoints of w_““; and
tiff define the proximal and distal ends of the E ribosomal
region (Hilliker and Appels, 1982). In(1)w‘n4 moves the
heterochromatin that normally lies distal to the rDNA to a
point near the g locus at the tip of the euchromatin; it
leaves the bulk of the ribosomal cistrons near the centromere.
In(1)w“‘51b moves most of the rDNA to the tip but leaves a very
small portion near the centromere. An inversion chromosome,
In(1)wm““wmm, bearing both nucleolus organizers was used as a
target for Egg-induced recombination. The two ends of this
parental chromosome were separated by a single crossover with
a deleted chromosome, In(1)w“mEMWR, to provide chromosomes

bearing the parental rDNA arrays (Robbins, unpublished).

II) SEPARATION OF RECOMBINANT NO'§

The result of hairpin exchange in In(1)w“"5“”“w‘“4R is a
normal order chromosome bearing two recombinant rDNA arrays,
Dp(1;1)w“‘51bwm4. These recombinant arrays are separated by a
crossover between Dp(1zl)wm51bw“l4 and Df(1)X1 which results in
the production of a normal E chromosome and a Tp(1;l);g‘”5“’wm4
chromosome (Fig. 9); each bearing a single nucleolus

organizer. Eleven pairs of recombinant NO's were generated

30

/n(1)wm51bLWm4FI’

23$

 

\’
«I,
.3 3.
- 3’ ’9. -.

 

V
/n(1)wm4 ? copies

 

? COpieS WWW/7757b

 

Figure 8 - Separation of parental NO's.

32

   

 

Dp(1,'1) Wm51 Wm4
Sgsgzfi
Df(1)X1

\/

 

Tp(1;1)wm57wm4, DF(1)X1

? copies

 

0

Normal X * ? copies

Figure 9 - Separation of recombinant NO's.

33
from the eleven Dp(1:1)wm5‘“wMR reinversions.

It should be noted that all the chromosomes used have
been kept in stock for several years with no sign of
breakdown. Swanson (1987) has observed each of these stocks
for at least. twelve: generations and. has never seen. any

products of spontaneous breakdown.

III) DNA DOT BLOTS

Multiple samples of genomic DNA are spotted next to each
other on a single filter in dots of uniform diameter. For
quantitative analysis, known amounts of DNA are applied. To
evaluate the extent of hybridization of the probe, a standard
consisting of a dilution series of DNA is applied in an
identical way to the same filter. The procedure binds samples
quickly so that many samples can be handled at once. rDNA is
readily detected in a spot containing as little as 50 ng of
total DNA. Dot blots do not distinguish the number and size of
the molecules hybridizing, so the hybridization "signal" is
the sum of all sequences hybridizing to the probe under the
conditions used. A general discussion of dot blot techniques
may be found in Hames and Higgins (1985).

Filters are treated with high concentrations of salt
prior to binding of nucleic acid. This both improves the
efficiency of binding and helps to keep the diameter of the
dot small. The salt solution most commonly used is 20x SSC (1

x SSC is 0.15M NaCl + 0.015M trisodium citrate, pH 7.0). For

34
the dot blots, DNA was covalently bound to the membrane by UV
irradiation (Stratalinker UV Crosslinker, 1800). It is
important to note that the filter must not at any stage be
handled with bare bands. Grease from the fingers will result
in poor binding of nucleic acids and high backgrounds.
Therefore disposable plastic gloves must be worn at all

stages.

IV) SIN§LE-INSECT SQUASH-BLOT

Nitrocellulose filters are first treated with high salt
solution, prior to squashing. This both improves the
efficiency of binding and helps keep the diameter of the
squash small. Flies, frozen at -72°C for a brief time, are
placed next to each other in the wells, squashed, under
vacuum, using a teflon dowel (6 to 8 rotations of rod). The
filter is then air dried at room temperature for 5 minutes,
denatured and neutralized. The filter is then either baked at
EHPC for 2 hours or UV fixed. To digest the proteins and other
debris, the filter is treated with chitinase in 10 ml citrate,
pH 5.5 for 18 hours and then treated with proteinase K at 38%:
for 2 hours. It is important to note that nitrocellulose
membranes cannot be readily stripped and reprobed. Note also

that disposable gloves must be worn at all stages.

35

V) RADIOLABELING OF DNA PROB§§
The DNA probes used in this study included: (1) an rDNA
probe containing a 300 bp sequence flanking the T1/T2
insertion sites in the 28S subunit. This fragment is present
once in each rDNA repeat. It has been cloned into the 2.7 kb
pUC18 vector (Jakubcak, personal communication) and (2) a
probe.containing the single copy Q.:melanogaster urate oxidase

gene (U0) (Wallrath et al., 1990).

VI) PROBE-LABELLINQgAND HYBRIDIZATION

rDNA and urate oxidase (UO) probes were labelled using
the oligonucleotide labelling method of Feinberg and
Vogelstein (1982) and labelled. probe. was separated from
unincorporated nucleotide by column chromatography using
Selphadex G-50.

Pre-hybridization is carried out overnight at 42°C in
heat-sealed bags with 10 mls of pre-hybridization solution
(50% formamide; 0.5 mg/ml alkali-sheared salmon sperm DNA; 1x
pre-hybridization.stock). For measuring'rDNA_copy'numbers, 10-
fold excess of unlabelled copies of the 32P—labelled probing
sequence and short hybridization times are used to ensure
appropriate kinetics. For the rDNA probe, hybridization is
carried out at 42%: in heat-sealed bags with 1.06 dpm/ml of
probe in hybridization solution ( 1x prehybridization stock;
50% formamide; 0.2 mg/ml alkali-sheared salmon spernlDNA). The

presence of 10x Denhardt's solution and denatured DNA in the

36
pre-hybridization and hybridization media, as well as the
washing under strigent.conditions reduce the background on the
filter resulting from non-specific hybridization.

After hybridization, the filters are washed twice with
2x SSC + 0.05% Sarkosyl + 0.02% sodium phosphate at room
temperature, followed by three 15 minute washes at 50%:anui
one 15 minute wash at 60°C in 0.1x SSC + 0.05% sarkosyl +
0.02% sodium phosphate. The filter is then wrapped in plastic
wrap to prevent drying, autoradiographed and counted using a
Betascan blot analyzer.

Hybridization of the single-copy urate oxidase gene was
used as an internal control. rDNA probe must be removed from
the hybond-N nylon membranes by incubating the blots at 45%:
for 30 minutes in 0.4M NaOH and for another 30 minutes in 0.1x
SSC + 0.1% SDS + 0.2M tris-HCl, pH 7.5. After this, the same
protocol of pre-hybridization, hybridization and washing
described above is used for the U0 probe with the exception
that hybridization is carried out for a longer time in the
presence of 10% Dextran Sulfate to ensure appropriate

kinetics.

VII) Hybridization kinetics

Filter hybridization depends on two processes, diffusion
of the probe to the filter and hybridization at the filter. It
is thought that at low concentration of filter-bound nucleic

acid sequences, the hybridization reaction itself is the rate

37

limiting step, whereas at high concentration of filter-bound
nucleic acid sequences, hybridization is so fast that the
solution surrounding the filter becomes depleted of probe and
the overall reaction is then limited by diffusion of the probe
to the filter. Increasing the concentration of the probe in
solution will increase the initial rate of hybridization at
the filter, and the proportion of the filter-bound sequences
in duplex will increase. Thus, for a multi—copy sequence such
as rDNA, a high probe concentration and short hybridization
times yield a measure of bound sequence. For the single-copy
UO gene, in contrast, high specific activity and long
hybridization times are appropriate because probe
concentration cannot be seriously depleted during the course
of the reaction. For the rDNA, therefore, we used a 10-fold
excess of un-labelled probe sequence and short hybridizations.
For U0, we used only labelled probe and longer hybridizations.

The validity of these conditions can be assessed by
examining the linearity and extent of hybridization with

variation of the amount of bound DNA.

VIII) Calculation of 10x eggaaa of probe

In Drosophila melanoqaster, there is approximately 400ng
of DNA in a single fly (Robbins, personal communication). The
rDNA represents about 2% of total DNA or 8ng; rDNA= (% DNA in
the:§,chromosome= 20%)x(% of heterochromatin in the X: 30%)x(%

of rDNA in heterochromatin= 30%). The total fragment DNA is

38
determined by the fragment size (= 400bp) divided by the rDNA
repeat length (= 11.5kb). Therefore there is approximately

3.3% (= 0.25ng) of fragment DNA in a single fly.

10x excess of probe corresponds, therefore, to 10 x 0.25
ng or 2.5ng of fragment DNA per fly or 6.25 x 10'3ng of
fragment DNA per ng of DNA.

The volume of hybridization solution per unit area of
membrane required for running a good reaction is 0.05 ml/cm2
and the membrane size is 100 cm2(8.5 cm,x 11 cmm). Therefore,
5 ml of hybridization solution are used to hybridize a single
membrane.

I normally use 1.0 x lwscpm/ml to do the hybridization,
corresponding to 5.0 x 106 ‘total counts in 5 ml of
hybridization solution. Once the probe is prepared and total
counts are determined, the volume of probe needed for a
hybridization reaction is determined as follows:

volume of probe needed = (5.0 x ldfl(total volume of probe)
Total counts of the probe

I3) COUNTINQ AND CALCULATIONS
1) - Counting

A betascope (Betagen corp., model 603) was used to
quantify the relative amount of 3JP present in each DNA dot.

Counting of the filter in the betascope for four hours using

the rDNA probe and for 18 hours using the single copy U0 probe

39

is sufficient to give intense signals.

2) - Calculations

A) Subtracting the background - the Betascope image is
divided into small 9.2 mm squares and small 9.2 mm diameter
circles around every DNA dot. The number of counts present in
each circle is subtracted from that present in the
corresponding square and the result is divided by (Area of
square - Area of circle) to determine the amount of background
radioactivity per unit area. This value is then multiplied by
the area of the circle and the result is subtracted from the
number of counts in that circle to determine the net counts
for the corresponding DNA dot.

B) Determination of the relative copy number — The gene
copy number for a particular genotype is measured relative to
our standard genotype, Ore—R. In the graphs, net counts for
both probes are plotted as a function of DNA amount for each
genotype tested and the slopes for both graphs are determined
by linear regression. Let these slopes be 81 and 82 for rDNA
and U0, respectively for the genotype tested and Scl and Sc2
for rDNA and U0 of Ore-R. The relative copy number is,

therefore: (Scl/Sc2)x(SZ/Sl)

 

PRELIMINARY RESULTS

I - S UASH-BLOT

In order to establish a technology for rapidly
determining rDNA copy number using single-insect squash-blots,
a number of different parameters have been examined. Here, I

present some preliminary results for the following:

1) - Different ways of keeping the dots small and improving
the efficiency of binding

2) — Different ways of fixing DNA onto filter.

3) - Linearity

1) - Improving the efficiency of binding and keeping the dots
small:

Treating the membranes with high salt, prior to DNA
application, improves the efficiency of binding and keeps the
dots small. For this experiment, the following concentrations

of salts have been applied to a number of filters:

a) Filter wet in 10 ml of H53 followed by 10 ml of 20x SSC

b) - Filter treated with 10 nfl.}50 followed by 10 ml of 10x
SSC

c) - Flies directly squashed in 5M NaOH with no pre-treatment

of membrane

d) - No treatment: Filter dry

40

 

41

MALES FEMALES: MALES FEMALES
,fl. '2 H   0..., , ,

«‘4' 5 . curt-«moo
eofirogiflogofi
r. I a": * 54”“ 9.06

SSS X02

J'- a" Q 90
5143’: 3 Vin-.2: 5 «31,993
“"5": - QQOQJOOQ
flmkwéfiflfifla

BAKED UV FIXED A

 

Figure 10 - Hybridization of genomic DNA from MAC)
males (2 NO's) and C(1)RM/_Q females (one NO) squashed on
nitrocellulose filter and probed with rDNA. Filter was cut in
half and each half was divided into two portions. One portion
was treated with 20xSSC prior to squashing and the other
portion was kept dry. One half was then baked at 86ktfor 90

minutes and the other half was UV fixed.

42

IWALES

1130

N210“

 

 

Figure 11 - Hybridization of genomic DNA from squashed
y: m flies probed with rDNA sequences. Prior to squashing,
filter was either treated with :90 (line a) 10x SSC; NaOH or

kept dry.

 

43
Techniques b and c gave too much variation. and the DNA
was spread out (Fig. 11). Filters treated with both techniques
a and d gave better results (Figures 10 and 11). Technique d
did not, however, give the same results for all the membranes

treated (Figures 10 and 11).

2) - Different ways of fixing DNA onto filter:

Two techniques have been tried: baking the membrane at
EHPC for one hour 30 minutes versus fixing DNA onto filters by
UV exposure. Several membranes were made, and each was cut in
half. One half was baked for 90 minutes, and the other half
was UV fixed (Fig. 10). In all cases, the results showed that
the UV exposure gives equal or better results than the baking

technique (Fig. 10).

3),- Linearity

For quantitative analysis, it is important to ensure that
the proportion of the probe hybridizing increases linearly
with DNA amount. For the squash-blots, visual comparison of
the intensity of hybridization signals on an autoradiogram
indicates that.we have not achieved this. Fig. 12 shows squash
blots of genomic DNA from a single fly (line a), two flies
(line b), three flies (line c) and four flies (line d). We
expect that the autoradiographic signals would increase from
one line to the next giving a ratio of 1:2:3z4. Fig. 128, for

example, shows that line a (a single fly) gives as intense

 

44

Figure 12 - Test for linearity.

A): Squash blot rDNA hybridization of homozygous y flies (two
NO's); and IN(1)sc“sc“/yl3_S and C(1)DX/_X (one NO)

B): Squash blot rDNA hybridization of M/Q (2 NO's) and
C(1)RM/Q (one NO). Single flies (line a), two flies (line b),

three flies (line c) and four flies (line d).

45

adeade

 

ClDX/X

1!!

B)

C“ )RM/Q FEMALES

MALES

YSX'YL/O

 

 

 

46

signals as line b (two flies) or line c (three flies); and in
Figure 12A, line a (one fly) and line c (three flies) give
more intense signals than line b (two flies) and line d (four
flies).

It is also important to note that, although the filter was
treated with high salt solution (Figure 12B) which should keep
the diameter of the spots small (see section 1), the DNA was
spread out. These inconsistencies led us to switch to DNA dot

blots.

II) DNA DOT BLOTS

 

The following parameters and comparisons have been tested
using DNA dot blots:
1) Different ways of denaturing the DNA
2) The time period of pre-hybridization

3) The time period of hybridization

1) - Different ways of denaturing the DNA

Two techniques have been tried: (1) heating the DNA at
75% for 20 minutes and application to the filter followed by
denaturing and neutralizing in 0.5M NaOH; 1.5 M NaCl and in
0.5M Tris, pH 8; 1.5M NaCl, respectively (Fig. 13A) and (2)
denaturing the DNA by heat only (Figure 13B). Although both
techniques give strong signals, the first technique appears to

be more efficient.

 

 

47

Figure 13 - Denaturation procedure and pre-hybridization time.
Ore-R.DNA.blots denatured.by heat only (A) or alkali-denatured
(B),were pre-hybridized for 1/2 hour, 2 hours, or 18 hours.

X

pA—56 (rDNA) probe

+ UO probe, —-- = linear regression, y = 0

 

§§§§§

NEl’ COUNTS

Igggguasaz

48

Oregon-R DNA, UO vs. pA-56, 100-400ng

alkali, 1/2 hr. prehybridization

 

§ §

7

§

 

 

_.<

B

 

 

alkali. 2 hr. prehybridization

 

 

 

 

alkali. 18 hr. pre-nybridizanoh

 

 

 

)0

NET COUNTS

heat. 1/2 hr. pre-hybridizatlon

 

 

 

 

 

x /
(I) /
g /-
x
8 / x
[G // - ‘“
Z / “fl __ *
6" ,. »— "‘"
S. m5
'so 200 250 500 use «,0
no DNA
heat. 2 hr. [Dre-hybridization
7000 /J
0000 //
"/
sooo ’//-"4
4000 ‘,-//
3000 ./1 ‘
--’//
2000 / ‘
4
1000 ,
‘1 )0 150 250 350 400
ng DNA
heal, 18 hr. prehybridization
eooo //'
M ”'II
an //fi'
5 . f,»
3 woo -
8 x”.-
m aooo ’/,-'
z ,-/ -
2000 D/ :, .-
;.
woo
‘i )o ‘50 200 300 350 am

 

 

 

 

 

 

 

49

2) - Time pariod of pre-hybridization

In the pre-hybridization step, the filter was incubated
at 42°C for 1/2 hour; 2 hours or 18 hours in a solution which
is designed. to jpre-coat. all the sites that. would, non-
specifically, bind the probe. The results are shown in fig.13.
In all cases, strong signals are observed with both the rDNA
probe and the U0 probe. With half hour pre-hybridization, not
only do we get strong signals, but the amount hybridization is

proportionate DNA amount.

3Li- Time period of hybridization

Hybridization with the rDNA probe was carried out at 42%:
for one; two; three or four hours (Fig. 14). Since the initial
rate of reaction is proportionate to the concentration of
bound DNA, we want to use the shortest hybridization time that
gives adequate signal. Long hybridization times may result in
depletion of the probe and a non-linear response. The results
shown in Fig. 14 suggest that one hour hybridization with the
rDNA probe is sufficient and that the signal varies
proportionally with bound DNA concentration. Prolonged
incubation ( Fig. 14) does not necessarily increase the extent
of hybridization because:
1) more and more probe reassociates
2) the probe concentration is reduced as it hybridizes to the

bound DNA.

50

Figure 14 - Ore-R DNA blots hybridized with rDNA probe for

one, two, three or four hours.

 

51

NET COUNTS

NET COUNTS

d IOCD I<wm_O_N>dOZ

 

 

 

dO-t

ALAL
v

 

   

:8.
88
88.
88.
88.
88...
o..sur§.moma_§_§.&o.
802)
w IOCD I<®D_O_N>._1_OZ
S. .
do
c
Q
:0, u.
m .
w w
m 9
“5
NA

 

. NET COUNTS

NET COUNTS

N IOCD I<®D_O_N>.:OZ

 

 

 

 

 

 

 

 

s3“
9“
3.8 .822... (.5. A... 2.... a... .8.
802)
A IOCD I<wm=u_N>dOZ
M . l
m
m

 

 

 

 

 

 

52
For the single copy UO probe, hybridization was carried
out at 42%: for four hours or 18 hours in the presence of
Dextran sulfate. Fig. 15 shows that 18 hour hybridization is
more efficient than 4 hours, while the preceding graphs
(fig.13) showed that the extent of reaction remains

proportionate to DNA amount even at this longer time.

53

4 HOURS ) OVERNIGHT

a); 9:.

 

IOONG

 

 

 

 

208 N0

 

 

 

388 ND

43.x

 

: 499 No

' see N5

 

688 N6

826 “i

 

Figure 15 - Ore-R DNA dot blot hybridized with 00 probe for

four hours or 18 hours.

CONCLUSION

We wished to find a convenient method for determining
rDNA copy number. Compared to other techniques, i.e. solution
hybridization, filter hybridization or single—insect squash—
blots, DNA dot blots were found to be a simple and sensitive
method. After testing a number of different parameters that
affect DNA binding and the hybridization reaction, the
following protocol has been established:

1) Float a sheet of nylon and two sheets of 3MM paper on
water taking care not to trap air bubbles underneath. When
one side is wet, immerse the membranes completely to wet
the other side.

2) Transfer the filter and the 3MM papers to a dish
containing 20X SSC. Leave for 10 minutes.

3) Dry at room temperature until completely dry

4) Dissolve DNA in TE (0.01M Tris, 0.001M Na2 EDTA) at a
concentration 0f 5 ug/ml. Heat these samples to boiling
for 10 minutes. Adjust each sample to a final
concentration of 2.5M NaCl by adding an equal volume of
5M NaCl.

5) Add samples of 50ng, 100ng, 150ng, 200ng, 250ng, 300ng,
350ng, 400ng to wells of Millipore blot apparatus, under
vacuum.

6) Air dry for 5 minutes.

7) Denature in 1.5M NaCl; 0.5M NaOH.

54

8)

9)

10)

11

v

12

v

13)

14

V

15)

55
Neutralize in 1.5M NaCl; 0.5M Tris—HCl, pH 7.2; 0.001M
Na2 EDTA.
Air dry for 5 to 10 minutes
Transfer filter to a Whatman 3MM paper saturated with 10X
SSC and expose to UV light for DNA fixing.
Pre—hybridize for 1/2 hour at 42°C in heat—sealed bags
with 10 mls of pre-hybridization solution (50% formamide;
0.5 mg/ml alkali-sheared salmon sperm DNA; 1X pre—
hybridization stock).
Hybridize for one hour with rDNA probe or 18 hours with U0
probe at 42°C in heat-sealed bags with 106 dpm/ml of probe
in hybridization solution (1X pre-hybridization stock; 50%
formamide; 0.2 mg/ml alkali—sheared salmon sperm DNA). The
rDNA hybridization mix has cold probing sequence added,
while the U0 mix has 10% dextran sulfate added
Wash filter twice with 2X SSC + 0.05% sarkosyl + 0.02%
sodium pyrophosphate at room temperature followed by
three 15 minute washes at 5Tb and one 15 minute wash at
6Tb in 0.1x SSC + 0.05% sarkosyl + 0.02% sodium
pyrophosphate.
Wrap filter in plastic wrap to prevent drying.

Autoradiograph and count using Betascan.

BIBLIOGRAPHY

Arnheim, N. 1983. Concerted evolution of multigene families.
IN: Evolution of genes and proteins, M. Nei & R.K. Koehn. eds.
(Sunderland, Mass., Sinauer Assoc.), pp.33-61.

Coen, E. S., J. M. Thoday and G. Dover. 1982. Rate of turnover
of structural variants in the rDNA gene family of Q.
melanogaster. Nature 295: 564-5568.

Dover, G. 1982. Molecular drive: a cohesive mode of species
evolution. Nature 299: 111-117.

Hames B. D. and S. J. Higgins eds., 1985. Nuclei acid
hybridization: a practical approach. IRL Press Ltd Oxford: 73-
112.

Hilliker, A. J., R. Appels and J. Schalet. 1980. the genetic
analysis of Q. melanogaster heterochromatin. Cell 21: 607-619.

Indik, K. and.K. D. Tartof. 1980. Long spacers among ribosomal
genes of Q. melanogaster. Nature 284: 477-479.

Kidd, S. J. and D. M. Glover. 1981. Q. melanogaster ribosomal
DNA containing type II insertions is variably transcribed in
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