Genetica78: 63-72, 1989
© KluwerAcademicPublishers, Dordrecht - Printedin the Netherlands
63
Taxonomic identification of Drosophila nasuta subgroup strains by glue
protein analysis
S. R. Ramesh 1 & W.-E. Kalisch 2
Institut fiir Genetik, Medizinische Fakultiit, Ruhr-Universitiit Bochum, Postfach 102148, D-4630 Bochum
1, Federal Republic of Germany
1Permanent address: Dept. of P.G. Studies & Research in Zoology, University of Mysore, Manasagangotri,
Mysore-570 006, India
2To whom all correspondence may be addressed
Received15.1.1988
Acceptedin revisedform 14.9.1988
Abstract
Protein fractions of salivary glands were analyzed from 30 wildtype strains of eight species belonging to the
Drosophila nasuta subgroup by SDS-polyacrylamide gel electrophoresis. The electrophoretic patterns indicated several prominent bands which could be shown to represent the major glue protein fractions. The glue protein
fractions are species-specific as well as wildtype strain-specific. Wildtype strain specificities are characterized
by variations of the species-specific patterns. The patterns of the different wildtypes, species, and hybrids were
used for taxonomic identification within the nasuta subgroup, in which the females are morphologically indistinguishable and the males differ only by the markings of their frons. The hybrids provide evidence for
gonosomal as well as autosomal linkage of individual genes coding for the major glue protein fractions.
Introduction
The nasuta subgroup of the immigrans species group
of Drosophila has been studied considerably from
the genetic and evolutionary points of view. In several cases the dose relationship of the members allows
for the obtainment of fertile hybrid progeny (cf:
Ramesh & Rajasekarasetty, 1980; Ramachandra &
Ranganath, 1986).
The Drosophila nasuta subgroup is, however, an
assemblage of morphologically almost identical
species. Females of this subgroup are morphologically indistinguishable, while the males have silvery
markings on the frons, the extent of which varies
(Nirmala & Krishnamurthy, 1972). Taxonomic identification of species obtained in the wild as well as
those from population genetic studies have so far
been based on cytological investigation (metaphase
plates, polytene chromosomes), or hybridization
studies (Ramachandra & Ranganath, 1986).
In this paper we describe a simple procedure to
identify different wildtype strains of the same species, different species, and interspecific hybrids
through their major glue protein fractions. The technique is based on SDS-polyacrylamide gel electrophoresis of the salivary gland proteins. We discuss
probable reasons for the wildtype strain-specific and
species-specific patterns together with evolutionary
aspects from data in the literature.
Material and methods
Stocks
The Drosophila melanogaster wildtype (Berlin)
strain has been used in our lab for about twenty
years, the D. nasuta nasuta strain (Mysore I in Table
64
1) was caught in Coorg, Karnataka, India and has
been used and maintained in the lab for the past ten
years. The other Drosophila strains of the nasuta
subgroup were obtained from the National
Drosophila Species Resource Center, Bowling
Green, Ohio, U.S.A. Further details regarding the
different wildtype strains of the species used in the
present investigation as well as kD-values of their
major glue protein fractions are given in Table 1.
Standard cornmeal food was used together with extra baker's yeast for the feeding of the larvae. Cultures were maintained at 22+1 °C.
Table 1. List of Drosophila stocks used in the experiments.
Nr a
15112-1781
15112-1781.1
15112-1781.2
15112-1781.6
15112-1751
15112-1751.1
15112-1751.2
15112-1751.4
15112-1751.5
15112-1761.1
15112-1771
15112-1771.1
15112-1771.2
15112-1771.3
15112-1771.4
15112-1801
15112-1831
15112-1831.1
15112-1831.2
15112-1831.3
15112-1831.4
15112-1821
15112-1821.1
15112-1821.5
15112-1821.6
15112-1821.7
15112-1811
15112-1811.1
15112-1811.3
15112-1811.5
15112-1811.7
Species
D. melanogaster
D. n. nasuta
D . n . nasuta
D. n. nasuta
D. n. nasuta
D. n. nasuta
D. n. albomicans
D. n. albomicans
D.
D.
D.
D.
K.
D.
D.
n. albomicans
n. albomicans
n. albomicans
n. kepulauana
kohkoa
kohkoa
kohkoa
D. kohkoa
D. kohkoa
D. pulaua
D. s. sulfurigaster
D. s. sulfurigaster
D . s . sulfurigaster
D. s. sulfurigaster
D. s. sulfurigaster
D.
D.
D.
D.
D.
s.
s.
s.
s.
s.
bilimbata
bilimbata
bilimbata
bilimbata
bilimbata
D.
D.
D.
D.
D.
s.
s.
s.
s.
s.
albostrigata
albostrigata
albostrigata
albostrigata
albostrigata
Original collection locality
of wildtype strains b
Berlin, Germany
Coorg, India (Mysore I)
Mysore, India (Mysore II)
Seychelles Is., Africa
Seychelles Is., Africa
Mombasa, Kenya, Africa
Okinawa, Japan
Alishan, Taiwan
Makung, Peng Hu Is., Pescadores
Bon Chakkrarat, Thailand
Komi, Ishigaki Is., Japan
Semongok For. Res., Sarawak, Malaysia
Bon Chakkrarat, Thailand
Sarawak, Malaysia
Mount Makiling, Luzon, Philippines
Samut Songkhram, Thailand
Rizal, Philippines
Semongok For. Res., Sarawak, Malaysia
Queensland, Australia
Kavieng, New Ireland
Wau, New Guinea
Papua, New Guinea
Kavieng, New Ireland
Tantalus, Oahu, Hawaii
Pago Pago, Tutuila, American Samoa
Tongatapu, Tonga Islands
Nadarivatu, Viti Levu, Fiji
Savusavuitangga, Vahua Levu, Fiji
Kuala Lumpur, Malaysia
Ari Ksatr, Cambodia
Semongok For. Res., Sarawak, Malaysia
Brunei, Borneo
Singapore, Indonesia
kD-valuescof protein fractions
in domains:
II
III
43.0
43.0
40.0
40.0
40.0
35.0
37.0
35.0
40.0
37.0
30.0
35.0
33.0
36.0
35.0
27.0 d
28.0
31.5
34.0
32.5
31.5
34.0
34.0
34.0
34.0
34.0
34.0
28.5; 27.0 d
26.0d
34.0
28.5; 28.0 d
34.0
30.0;
31.0;
26.0;
26.5;
27.5;
25.5;
24.0;
25,5;
18.5;
27.0;
23.0;
20.0;
25.0;
28.0;
21.0;
25.0;
25.5;
22.0;
25.5;
24.5;
24.0;
25.5;
21.5;
25.5;
25.5;
25.5;
25.5;
26.5;
25.0
25.0;
25.0;
25.0;
IV
28.0
30.0
25.0
25.5
25.5
23.0
23.0
23.0
18.0
25.5
22.0
19.0
24.0
27.0
19.5
24.0
23.0
21.0
24.5
23.0
22.0
24.5
20.0
24.0
24.0
24.0
24.0
24.5
23.0
23.0
23.0
14.0
14.5;
14.0
14.0
14.5;
14.0
14,0
14.0
14.0
14.0
14.0
14.0;
14.0;
14.0;
14.0;
14.0;
14.0;
14.0;
13.5
14.0;
14.0;
13.5
14.0;
14.0;
14.0;
14.0;
14.0;
14.0
14.0
14.0
14.5
14.0
14.0
14.0
13.0
13.0
13.0
13.0
13.0
13.5
13.5
13.5
13.5
13.0
13.0
13.5
13.0
13.0
a Code numbers from the Drosophila Species Stock List (1984) of the National Drosophila Species Resource Center, Bowling Green
State University, Bowling Green, Ohio, U.S.A.
b For more details see Wilson et aL (1969).
c Approximate kD-values of the dot-labeled bands in Figs. 2 - 8 , according to the present results from several experiments with different
protein extracts.
d Major glue protein fractions which could belong to the adjacent domain on the basis of their kD-values.
65
Preparation of samples
Mid third-instar larvae were used in all investigations
in order to rule out the possibility of any age-specific
variation in the pattern of glue protein (Ramesh &
Kalisch, 1988). Larvae were washed and dissected in
a medium (Ashburner, 1970) containing 0.03 M
Na2PO 4 buffer (pH 6.8), 0.04 M KCI, 0.011 M
NaC1, 0.003 M CaCI2, and 0.021 M MgCI2 at
20+_1 °C. Glands were transferred for 20 min to a
1.5 ml Eppendorf tube filled with cold 10% TCA (=
Trichloracetic acid). The TCA was pipetted from the
Eppendorf tube and the glands were washed (each
time for 20 min) with 95°7o ethanol, as well as with
a mixture of methanol and chloroform (l:l) by pipetting it several times using a Pasteur pipette. The tissue was finally allowed to dry at 37 °C. 30 ~l (for single pair extracts) or 50/xl (for multiple pair extracts)
of a sample buffer [0.0625 M Tris-HCl (pH 6.8), 1%
SDS, 1% /3-mercaptoethanol, 10% Glycerol, and
0.001% Bromophenol blue] was added to the dried
tissue. The Eppendorf tubes were tightly closed and
the contents were heated in a boiling water bath for
10 min. Tubes were then cooled to room temperature
and centrifuged for 5 min at 1000 rpm. These extracts could be stored at - 2 0 ° C for a few days.
Electrophoresis
20 #I samples were loaded in each slot on a 13.7°70
SDS-PAAG (with 0.8o7o bisacrylamide) of 1 mm
thickness. Using a 0.05 M Tris, 0.384 M glycine
buffer (pH 8.3) containing 0.1070 SDS as electrode
(tray) buffer, the electrophoresis was performed at
165 volts for 3½ h at 20+_1°C, until the
Bromophenol blue migrated to 8 cm in the separation gel. The gel was treated overnight in a prestaining solution [50o7o TCA and isopropanol (hl)],
stained in 0.15 % Coomassie brilliant blue R-250 for
2 h, and destained (25% methanol and 7.5% acetic
acid). For separating closely migrating double
bands, as for example the 30 kD and the 28 kD
bands in D. n. nasuta (compare Fig. I and 2) we used
diluted running buffer (1"1 with distilled water) and
15/~1 of the sample per slot. Polyacrylamide gels
were selected instead of starch gels as supporting
material for electrophoresis, because of the higher
power of resolution, the possibility to change the
pore size by changing the monomer concentrations
and other advantages (Gordon, 1969).
Marker proteins
For calculation of kD-values we used the following
combination of marker proteins (electrophoretic
pattern in Fig. 9): Phosphorylase a (97 kD), Conalbumin (86 kD), Bovine serum Albumin (68 kD),
Ovalbumin (45 kD), Carbonic Anhydrase (29 kD),
Myoglobin from whale (14 kD), and Cytochrome C
(12.4 kD).
Results
Salivary gland protein fractions in Drosophila
nasuta and D. melanogaster
Patterns of the protein fractions from the salivary
glands of mid third-instar larvae were compared in
Drosophila nasuta nasuta and in D. melanogaster
using 13.7070 SDS-polyacrylamide gel electrophoresis (Fig. 1). Both patterns differ remarkably due to
the presence of at least five prominent bands in D.
n. nasuta (labeled: > 100 kD, 43 kD, 30 kD, 28 kD
and 14 kD; for separation of the 30 kD/28 kD double band see Fig. 2). In further studies (Ramesh &
Kalisch, 1987; 1988), we were able to show that these
prominent protein fractions belong to the major
fractions of glue proteins. We were also able to
demonstrate that besides the 30 kD/28 kD double
band, the prominent bands of > 100 kD and 43 kD
are composed of tightly neighbored multiple bands.
The total amount of glue proteins produced in D. n.
nasuta is roughly twice that in D. melanogaster when
estimated in relation to the total amount of proteins
including the glue proteins (Ramesh & Kalisch,
1988).
Comparison of the glue protein patterns in eight
members of the nasuta subgroup
Eight members belonging to the nasuta subgroup
66
Fig. 2. Salivarygland protein fractions in different members of
the nasuta subgroup: (A) Drosophila nasuta nasuta (Mysore I);
- (B) 1). n. albomicans (-1751; for code numbers see Table 1);
- (C) D. n. kepulauana (-1761.1); - (D) D. kohkoa (-1771);
- (E) D. pulaua (-1801); - (F) D. sulfurigaster sulfurigaster
(-1831.2); - (G)D.s. bilimbata(-1821.6); - (H)D. s. albostrigata [ - 1811;same strain as in Fig. 11Eand F]. Three pairs of salivary
Fig. 1. Patterns of salivary gland protein fractions in (A)
Drosophila melanogaster (wildtype strain Berlin) and (B)
Drosophila nasuta nasuta (wildtype strain Mysore I) in a 13.7°/0
SDS-polyacrylamidegel. Eight pairs of salivary glands of mid
third-instar larvae wereused for each protein extract. Coomassieblue staining, kD-valuesare givenfor the major glue protein fractions of D. n. nasuta.
were studied for their patterns of glue protein fractions. Figure 2 shows the patterns of total salivary
glands (together with the glue proteins in them) to
indicate the extent of species-specific differences of
glue as well as the differences of the cellular proteins
if any. Evidence from electrophoretic data of isolated saliva plugs showing that in all species the prominent bands represent glue protein fractions are given
elsewhere (Ramesh & Kalisch, 1988 and in preparation).
It is obvious from the patterns in Figure 2 that the
prominent glue protein fractions in different species
show homologies and can be grouped at least into
four domains: Domain-I band(s) [homologous with
the < 100 kD band(s) inD. n. nasuta] represent fractions o f more than 100 kD in all the different species
analyzed. Since on one hand, the glue protein fractions o f this domain represent very tight multiple
glands of mid third-instar larvae were used for each protein extract (in Figs. 2 - 8). I - IV indicatedomains of'homologous'protein fractions in the different species(seetext). Dot-labeledbands
(in Figs. 2-8) indicate glue protein fractions in domains II-IV
as far as analyzed and for which kD-valuesare given in Table 1.
Cf. Material and methods.
bands (two bands in D. n. nasuta; Ramesh & Kalisch,
1988) and show little variability on the 13.7% SDSpolyacrylamide gels of this study on the other, we
have not included the domain-I bands in the discussion of the present investigation. It is even obvious
from Figure 2, however, that there are differences between the domain-I bands in the individual species
of the n a s u t a group. Domain-II bands [homologous
with the 43 kD band(s) ofD. n. nasuta] vary between
one band in D. k o h k o a (Fig. 2D) and m a n y bands
in D. n. a l b o m i c a n s (Fig. 2B; for comparison see
also Figs. 4 and 5). D o m a i n - I l l band(s) [homologous with the 30 kD and 28 kD bands ofD. n. nasuta] exist mostly as two fractions, of which the one
with the higher mobility (with the lower kD-value)
is the more prominent one. Domain-IV band(s)
[homologous with the 14 kD band of D. n. nasuta]
are represented by one or two fractions. Bands of domain IV show little variation with regard to the kDvalues in different species and wildtypes (Figs. 3 - 8 ;
Table 1).
67
The species-specific characteristics of the patterns
of glue protein fractions are mainly represented by
bands of the domains II and III. Whether or not
bands of homologous domains in different species
represent protein fractions with 'homologous' functions in the glue molecules is so far uncertain. Furthermore, it is still uncertain what the differences are
between homologous bands on the molecular genetic level.
The glue protein patterns from different larvae belonging to the same wildtype and species are reproducible. To minimize differences based on gland
sizes, species-specific developments, and preparation of protein samples, however, we used more than
one pair of salivary glands in most of experiments
of the present study.
Fig. 3. Salivary gland protein fractions o f D. n. nasuta v, ildtx pc
Glue protein patterns in different wildtype stocks
From the comparative studies in D. melanogaster
one knows that there is a great variability in the
major glue protein patterns among different wildtype stocks (Beckendorf & Kafatos, 1976; Korge,
1977; Velissariou & Ashburner, 1980). Therefore, in
addition to the strains in Fig. 2, we analyzed several
wildtype strains of six species of the nasuta subgroup
(Table 1).
Figure 3 shows five different D. n. nasuta wildtype
strains. The characteristic nasuta pattern was found
in all the strains but they also showed minor differences. The 'homologous' bands of domain III are
slightly different in all of the wildtype patterns
depicted. In domain IV, only the Mysore II and the
Kenya strains show double bands. Furthermore, the
geographical relationship of the original collection
sites is obvious from the comparison of the domain
II bands. The two strains from southern India have
a greater similarity with each other than with the
three strains from Africa and vice versa.
In Figure 4 we have shown the patterns in five
different wildtype strains of D. n. albomicans.
Although a characteristic D. albomicans pattern is
presented by all of the wildtype strains, there is also
a lot of wildtype strain-specific variation. This could
be simply correlated, at least partially, to the fact
that D. albomicans has the highest number of bands
strains from different collections: (A) Mysore I, India [same strain
as in Fig. 1B and 2A]; - (B) Mysore II, India; - (C) Seychelles
Is. I, Africa; - (D) Seychelles Is. II, Africa; - (E) Mombasa,
Africa. Cf. legend of Figure 2.
in domains II and III of all species checked so far.
Figure 5 represents five different D. kohkoa wildtype strains. The two strains from Thailand (A and
D in Fig. 5) are only slightly different, whereas the
Fig. 4. D. n. albomicans wildtypes: (A) Okinawa, Japan [same
strain as in Fig. 2B]; s (B) Alishan, Taiwan; - (C) Makung, Peng
Hu Is., Pescadores; - (D) Bon Chakkrarat, Thailand; - (E)
Komi, Japan. Cf. legend of Figure 2. Unlabeled prominent bands
in domains II and III so far have not been analyzed.
68
Fig. 5. 1). k o h k o a wddtypc~; (A) BOLl Chakkarat, lhailand
[same strain as in Fig. 2D]; - (B) Sarawak, Malaysia; - (C)
Mount Makiling, Phillippines; - (D) Samut Songkhram,
Thailand; - (E) Rizal, Philippines. Cf. legend in Figure 2.
Fig. 7. D. s. bilimbata wildtypes: (A) Nadarivatu Viti Levu,
Fiji [same strain as in Fig. 2G]; - (B) Tantalos, Hawaii; - (C)
Pago Pago, Am. Samoa; - (D) Tongatapu, Tonga Islands; - (E)
Savusavuitangga, Fiji. Cf. Figure 2.
remaining three strains seem to have little in common as far as domains II and III are concerned. Figure 6 indicates characteristic differences in the mobility of similar patterns of the protein fractions in
domains II and III between D. s. sulfurigaster
strains. The D. s. bilimbata wildtype strains in Figure
7 show the highest degree of pattern similarity, even
though they are from different islands from all over
the Pacific. All of them, among minor differences
show one characteristic additional band between the
prominent bands of domains II and III.
The four wildtype strains of D. s. albostrigata in
Figure 8 can be divided into two groups: A 'typical'
albostrigata pattern with the characteristic narrow
set of major glue protein bands of domains II and
III (A and C in Fig. 8), and the remaining two strains
Fig. 6. D. s. sulfurigaster wildtypes: (A) Queensland, Australia;
- (B) Kavieng, New Ireland; - (C) Papua, New Guinea; - (D)
Kavieng, New Ireland, Cf. Figure 2.
Fig. 8. D. s. albostrigata wildtypes: (A) Ari Ksatr, Cambodia;
- (B) Semongok Forest Reserve, Sarawak; Malaysia; - ((2)
Brunei, Borneo; - (D) Singapore, Indonesia. Cf. Figure 2.
69
(B and D in Fig. 8) which are almost identical with
each other and look like the D. s. sulfurigaster
(Fig. 6) and D. s. bilimbata (Fig. 7) patterns with
slightly different mobilities, but are identical with
none of these. The differences found concern bands
in the overlapping regions of domains II and III as
well as in domain IV.
Identification o f hybrids and location o f genes
coding f o r major glue protein fractions
In interspecific hybrid larvae, one can determine the
genetic background of both parents by the characteristic pattern shown in Figures 2 - 8 . This technique could be used for taxonomic identification of
individual larvae. Experiments of this type could
also be of use in population genetic studies and
should even provide an easy means to examine
hybridization in nature. In the following, we give
three examples.
In Figure 9C, D. n. nasuta x D. n. albomicans
hybrid females show the full set of protein bands
from both parents (A and E). In connection with the
hybrid females shown in Figures 10D and liD, each
hybrid can be identified and characterized by the set
of its major glue protein fractions. The technique,
however, is limited to female hybrids because the
major glue protein fractions of domains II and III
are X-chromosomally linked in all the members of
the nasuta subgroup tested so far.
Evidence for the X-chromosomal linkage of genes
coding for the major glue protein fractions is given
in Figure 9 for D. n. nasuta and D. n. albomicans:
The patterns of protein fractions of one pair of salivary glands are shown for one D. n. nasuta male (A)
and one D. n. albomicans male (E) as well as for
reciprocal Fl-hybrid males (B and D) and a hybridfemale (C). In reciprocal crosses (Fig. 9) the Flhybrid males show, in comparison with Fl-hybrid
females, only the glue protein fractions of their
mothers. This involves the 43 kD, 30 kD, and 28 kD
bands in D. n. nasuta (arrows in Fig. 9B; compare
also Fig. 1B) and at least the 35.0 kD, 25.5 kD,
23 kD bands in D. n. albomicans (arrows in
Fig. 9D). Since the Fl-hybrid males receive their Xchromosome from their mothers, the X-chromo-
Fig. 9. Salivary gland protein fractions of D. n. n a s u t a (Mysore
I) x D. n. a l b o m i c a n s ( - 1 7 5 1 ) hybrids as well as evidence for Xchromosomal linkage of major glue protein fractions in D. n.
n a s u t a and D. n. a l b o m i c a n s : (A) male of D. n. nasuta; - (B)
male- and (C) female-hybrid from a D. n. n a s u t a mother and a
D. n. a l b o m i c a n s father; - (D)male-hybrid from a D. n. a l b o m i c a n s mother and a D. n. n a s u t a father; - (E) male of D. n. alb o m i c a n s . Each protein extract from one pair of salivary glands
from a mid third-instar larva. Arrow-labeled bands indicate
major glue protein fractions of both species which are coded by
X-chromosomal genes. (M) Marker proteins [listed in Material
and methods].
Fig. 10. Salivary gland protein fractions of D. s. s u l f u r i g a s t e r
( - 1831.2) x D. s. b i l i m b a t a ( - 1821.6) hybrids as well as evidence
for X-chromosomal linkage of the major glue protein fractions:
(A) male and (B) female of D. s. sulfurigaster, - (C) maleand (D) female-hybrid from a D. s. s u l f u r i g a s t e r mother and a
D. s. b i l i m b a t a father; - (E) male and (F) female ofD. s. b i l i m b a ta. Cf. Figure 9.
70
Salivarygland protein fractions in D. s. sulfurigaster
(-1831.2) x D. s. albostrigata (-1811) hybridsand evidencefor
X-chromosomal linkage of genes: (A) male and (B) female of
D. s. sulfurigaster; - (C) male-and (D) female-hybridfrom a D.
s. sulfurigaster motherand a D. s. albostrigata father; - (E) male
and (F) femaleof D. s. albostrigata. Cf. Figure9.
Fig. 11.
somal linkage of genes can be confirmed.
In Figure 10, comparable results are given for O.
s. s u l f u r i g a s t e r and D. s. b i l i m b a t a , i.e., the major
glue protein fractions of the domains II and III (labeled in Fig. 10) are coded by X-chromosomal linked
genes. In this case, the pattern of a reciprocal F lhybrid is not shown. Figure 11 shows evidence for the
X-chromosomal linkage of genes coding for bands
of domains II and III in D. s. s u l f u r i g a s t e r and D.
s. a l b o s t r i g a t a .
Discussion
The present pattern analysis of glue protein fractions
in the n a s u t a subgroup indicates that the 30 strains
as well as the hybrids tested so far, can be identified
individually by the technique used. It can not be excluded, however, that additional wildtype strains
may exist which are even more similar than the D.
s. b i l i r n b a t a strains (Fig. 7). In this case, our technique would have to be extended to the minor glue
and tissue protein fractions to be sensitive enough
for an individual identification. Also in those
hybrids in which protein fractions overlap in their
mobility with each other, it would be more difficult
to tell the genotypes of the parents apart. Nevertheless, our technique is, so far, easier to handle for taxonomic identification than any technique based on
cytological and/or morphological differences. Furthermore, our technique can even be used for the
identification of wildtype strains and hybrids.
The n a s u t a subgroup-specific pattern of the glue
protein fractions (domains I - I V ) with its speciesspecific differences on the one hand and the wildtype strain-specific variations on the other is of interest from the evolutionary and the molecular
genetic point of view. Species-specific differences include (besides quantitative differences) variations in
the number of fractions within the four domains
(Fig. 2) as well as variations of the electrophoretic
mobility of the fractions within each of the domains
(Fig. 3 - 8). The different number of 'homologous'
protein fractions in wildtype strains and in species
could be based upon chromosome mutations like
gene duplications, whereas the different mobilities
could be based among other possibilities on point
mutations. One knows that even the substitution of
a single amino acid can already significantly change
the mobility of a protein in a gel (Noel e t al., 1979).
However, so far little is known about the phylogenetic relationships between different members of the
n a s u t a subgroup on the basis of isoenzyme variations (Ramesh & Rajasekarasetty, 1980) and nothing
is known about the gene structure of the n a s u t a subgroup on the DNA level. In another study (Ramesh
& Kalisch, 1988) we were able to show that in D. n.
n a s u t a the prominent glue proteins of domains
I - III are PAS positive. So, the differences in the mobility of these protein fractions could also be based
on differences in the glycosylation of the protein
fractions.
Even though the 30 strains tested are different
from each other, it is surprising how similar the
general pattern of prominent glue protein fractions
(domains I - I V ) is within the n a s u t a subgroup
(Fig. 2). There are several other proteins in nature
which have been shown to be very similar in different
species, as for example the hemoglobin, insulin and
histone molecules. The need of conserving the essential function of these proteins has been obviously a
selecting factor during evolution, leading only to
71
minor changes between different species. In the case
of glue proteins, however, it is hard to believe that the
survival of the flies is dependent on a specific composition of glue, because completely different patterns have been reported in wildtype strains of D.
melanogaster (Beckendorf & Kafatos, 1976; Korge,
1977; Velissariou & Ashburner, 1980), in D. hydei
(Ramesh & Kalisch, unpublished), and in others
(Manousis, 1985). In this connection, one also has
to consider the possibility that the genome posesses
the ability to regulate differentially the rate of evolution of DNA sequences in different chromosomal locations (Martin & Meyerowitz, 1986).
Finally, we know from Figures 9-11 as well as
from a cytologic and formal genetic study (Ramesh
& Kalisch, 1988) that the X-chromosomal genes coding for fractions of domains II and III are tightly
linked and probably form a cluster of tandemly arranged genes within the huge puff of subdivision 10
(in the reference map of D. n. nasuta, published by
Ranganath & Krishnamurthy, 1974). This tandem
arrangement of the glue protein genes could probably indicate a situation similar to the location of the
glue polypeptides sgs-3, sgs-7, and sgs-8 of the 68C
puff in D. melanogaster (Garfinkel et al., 1983), i.e.,
a factor which conserved the basic pattern.
Results of the present study can be compared with
evolutionary genetic data from the literature. Wilson
et al. (1969) have shown that the three D. sulfurigaster species are more related with each other
than with the remaining species of the nasuta complex. Based on our data in Figure 2 this agrees with
the results in D. s. sulfurigaster and D. s. bilimbata,
but not with the results in D. s. albostrigata. However, the wildtype strains from Malaysia and Indonesia
(B and D in Fig. 8) in comparison with Figures 6 and
7 indicate that there is in fact a stronger relationship
between the three sulfurigaster species than to the
Cambodia strain (H in Fig. 2 and A in Fig. 8) or the
Borneo strain (C in Fig. 8). By this, the two albostrigata wildtype strains, which show the bilimbata
and sulfurigaster-like pattern (B and D in Fig. 8),
have to be seen as the more original ones, whereas
the two characteristic narrow-banded patterns (A
and C in Fig. 8) could be based on more recent evolutionary events.
Acknowledgements
S.R.R. is grateful to the Univ. Grants Commission,
New Delhi, India, for sponsoring and to the
Deutscher Akademischer Austauschdienst, FR Germany, for the award of a scholarship. This study is
part of a project (Ka 309/9-1) supported by the
Deutsche Forschungsgemeinschaft. The authors
wish to thank Mrs. Ch. Plehn for excellent technical
assistance and Mr. T. Whitmore for linguistic aid.
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