Drosophila Cytogenetics Protocols [Methods in Molec Bio 247] - D. Henderson (Humana, 2003) WW.pdf
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Methods in Molecular Biology
TM
VOLUME 247
Drosophila
Cytogenetics
Protocols
Drosophila
Cytogenetics
Protocols
Edited by
Daryl S. Henderson
Edited by
Daryl S. Henderson
Drosophila Chromosomes
1
1
The Chromosomes of Drosophila melanogaster
Daryl S. Henderson
1. Introduction
Drosophila
have two basic forms of chromosomes—mitotic and polytene—
that have vastly different morphologies and cellular roles. Polytene chromo-
somes are found in interphase nuclei of differentiated cells, being especially
prominent in certain tissues of the larva and adult ovary. They are produced by
repeated rounds of chromosome replication unhitched from nuclear division in
a process termed “endoreplication.” Among the largest and most familiar of
polytene chromosomes are those of the larval salivary gland, which can consist
of >2000 sister chromatids tightly aligned in register. Such scaled-up chromo-
somes permit production of large quantities of gene products in a narrow
developmental window. The highly compact mitotic chromosomes, found in
proliferating tissues (e.g., the larval central nervous system [CNS], imaginal
discs, ovaries, and testes), are genome-packaging vehicles that, in association
with the spindle apparatus, function to transmit complete copies of the genome
between mother and daughter nuclei. Meiotic chromosomes also can be cat-
egorized as mitotic chromosomes, and some of their unique properties are
touched on in Chapters 2–5 (for recent reviews,
see
refs.
1–3
).
Just as mitotic and polytene chromosomes serve different functions in the
fly, they are exploited by drosophilists for different purposes. The large size
and distinctive banding patterns of salivary gland polytene chromosomes—
Darlington likened them to “contorted earthworms”
(4)
—make them an excel-
lent material on which to locate genes and gene products
in situ
(
see
Chapters
13–15) or to finely map the breakpoints of chromosomal aberrations (
see
Chapters
12–13). The rise of
Drosophila melanogaster
to preeminence as an experimen-
tal organism owes much to these giant chromosomes. Mitotic chromosomes
are useful for investigating basic questions of mitotic and meiotic cell biology
From: Methods in Molecular Biology, vol. 247: Drosophila Cytogenetics Protocols
Edited by: D. S. Henderson © Humana Press Inc., Totowa, NJ
1
2
Henderson
(
see
Chapters 2–5, 17, and 18), including cytological studies of heterochroma-
tin (
see
Subheading 3.
and Chapters 16, 18, and 19).
This chapter provides an introduction to the chromosomes of
D. melanogaster
,
in both their mitotic and polytene forms. It begins with an outline of some of
the pioneering work in the field of
Drosophila
cytogenetics, focusing mainly
on achievements from the first half of the 20th century. More recent discover-
ies, flowing from advances in molecular biology, microscopy, probe technol-
ogy, and electronic imaging, are referred to later in the chapter and throughout
this volume.
2. Drosophila Cytogenetics: Early Milestones
Sutton’s 1903 landmark paper, “The Chromosomes in Heredity” (
5
;
see also
ref.
6
), in which he pointed out that the behavior of chromosomes in meiosis
parallels the observed patterns of inheritance of Mendelian traits, is considered
to mark the beginning of the field of cytogenetics (the actual term would be
coined years later). Before then, cytology with its focus on animals specimens,
and genetics, which consisted of breeding experiments involving mainly plants,
had been separate areas of inquiry
(6)
. In the practical melding of cytology and
genetics that soon followed,
D.
melanogaster
would be unrivaled in its funda-
mental contributions to the new field of cytogenetics, many of which are listed
in
Table 1
.
Ahead of her time and seldom acknowledged since, Nettie Stevens of Bryn
Mawr College, Pennsylvania
(34
,
35)
, was the first person to study chromo-
somes of
Drosophila
, beginning in the autumn of 1906
(7)
. Stevens, a codis-
coverer of chromosomal sex determination, was already an accomplished
cytologist when she worked the preceding summer at Cold Spring Harbor, NY,
examining chromosomes of cucumber beetles (
Diabrotica
spp.;
see
ref.
36
). It
is then and there that she likely obtained her first
Drosophila
specimens from
entomologist Frank Lutz. It was Lutz also, according to Kohler
(37)
, who prob-
ably introduced Morgan to
Drosophila
that same year. Attempts in Morgan’s
laboratory to identify
Drosophila
mutations did not begin in earnest until either
the fall of 1907 or 1908, and it was not until 1910 that the first unequivocal
mutants were actually found
(8
,
37)
. Thus, Stevens’ cytological studies of
D. melanogaster
(then called
D. ampelophila
) predate the first use of
Droso-
phila
for genetic analysis.
Stevens’ research was important because it helped substantiate Sutton’s
chromosome theory of heredity
(5)
. Indeed, she was far ahead of Morgan in
recognizing the significance of chromosomes
(34
,
35)
. For example, Stevens
discovered that the karyotypes of male and female
Drosophila
(and other
insects) differ at a single chromosome pair, from which she inferred a role for
such heteromorphic chromosomes in determining sex
(7
,
38)
, following
Drosophila Chromosomes
3
Table 1
Some Notable Achievements in Studies of Drosophila Chromosomes
1906
N. M. Stevens begins first ever study of
Drosophila
chromosomes;
observes heterochromosomes (X and Y) in males; observes somatic pair-
ing of homologous chromosomes (
7
; see text).
1910
T. H. Morgan discovers sex-linked inheritance; first assignment of a
specific gene (
white
) to a specific chromosome (the X)
(8)
.
1913
A. H. Sturtevant constructs the first genetic map (involving X-linked
genes)
(9)
.
1914, 1916
C. W. Metz builds on Stevens’ work; examines chromosomes in approx
80 species of Diptera, in gonads of both sexes, and in somatic tissues of
embryos, larvae and pupae
(10
,
11)
.
1916
C. B. Bridges proves chromosome theory of heredity through observa-
tions of nondisjunction
(12)
.
1916
H. J. Muller discovers crossover suppressors, later shown to be chromo-
some inversions, from which the concept of “balancer” chromosome is
derived
(13)
.
1917, 1919
C. B. Bridges describes the first chromosome deficiency, first chromo-
some duplication (inferred from genetic analysis)
(14
,
15)
.
1930
H. J. Muller discovers variegating mutations (“eversporting displace-
ments,” e.g., white-mottleds) resulting from chromosome rearrange-
ments
(16)
.
1930s
E. Heitz investigates
Drosophila
heterochromatin
(17–19)
.
1931
T. S. Painter discovers giant chromosomes in larval salivary glands and
demonstrates their usefulness for mapping
(20–25)
.
1934
B. P. Kaufmann publishes survey of chromosomes from various tissues
of
Drosophila
; reports that cells of the larval brain are most useful for
observing mitoses; presents extensive morphological description of
same
(26)
.
1935
C. B. Bridges devises map coordinate system for polytene chromosomes
(27)
.
1938
H. J. Muller and colleagues coin the term “telomere” to describe the
specialized ends of chromosomes
(28)
.
1940s
T. O. Caspersson undertakes first cytochemical studies using micros-
copy
(29)
.
1959
K. W. Cooper undertakes extensive cytological investigation of hetero-
chromatin, including morphological descriptions of the X and Y hetero-
chromatic elements
(30)
.
1969
M.-L. Pardue and J. G. Gall develop in situ hybridization method for
polytene chromosomes
(31)
.
1972
D. L. Lindsley et al. systematically analyze the genome using synthetic
duplications and deficiencies created in crosses of Y-autosome translo-
cation stocks
(32)
.
1977
G. T. Rudkin and B. D. Stollar demonstrate first FISH experiment
(33)
.
Note:
Some of these advances may be considered purely “genetic” rather than “cytogenetic,”
but they are included as historical reference points.
4
Henderson
McClung
(39)
. [
N.B.
: It was later established by Bridges that sex in
Droso-
phila
is determined by the ratio of X chromosomes to sets of autosomes
(40)
,
and not by the presence or absence of a Y chromosome, as in humans, for
example. The Y chromosome of
Drosophila
is essential for male fertility but
not for male
ness
.] Stevens was also the first to note the tendency of homolo-
gous chromosomes of Diptera to pair in diploid somatic cells (i.e., outside of
meiosis;
see
ref.
7
).
The methods Stevens used to study insect chromosomes are not so different
from the basic techniques we use today. She dissected testes and ovaries of
adult flies in physiological salt solution, transferred the tissues to a drop of
stain (acetocarmine) on a microscope slide, pressed the cover slip down to
break and spread the cells, and removed the excess stain by wicking with filter
paper. Of the nine dipteran species she studied, Stevens found the tissues of
Drosophila
to be the most difficult to work with, requiring her to examine an
inordinate number of specimens. She wrote,
While in Sarcophaga all the stages necessary for a description of the behavior of the
heterochromosomes of both sexes were found in the course of a few hours’ work on
perhaps ten or twelve preparations, satisfactory results in the case of Drosophila have
been obtained only after prolonged study extending over more than a year and involving
dissection of some two thousand individuals.
(7)
(See Chapters 2–5, and reduce the number of your
Drosophila
dissections to
Sarcophagan levels!) Despite such inauspicious beginnings, Stevens’ camera
lucida drawings of
Drosophila
prophase figures clearly show a complement of
eight chromosomes, with males having a heteromorphic pair (later designated
X and Y). Stevens concluded, “The general results of the nine species of flies
are the same; i.e., an unequal pair of heterochromosomes in the male leading to
dimorphism of the spermatozoa, and a corresponding equal pair in the female,
each equivalent to the larger heterochromosome of the male. . .”
(7)
. Unfortu-
nately, Stevens’ promising work on flies was abruptly stopped by the breast
cancer that claimed her life in 1912. It was left to Charles Metz
(10
,
11)
and
many others to build on Stevens’ discoveries (
see
Table 1
).
3. Mitotic Chromosomes
Diploid nuclei of wild-type
D. melanogaster
contain eight chromosomes
(2
n
=8) that can be seen most easily in squash preparations of the third instar
larval CNS (
see
Fig. 1
). The autosomal complement consists of two pairs of
large metacentric chromosomes, designated 2 and 3, and a pair of tiny, spheri-
cal fourth chromosomes. Chromosomes 2 and 3 appear morphologically very
similar after aceto-orcein or Giemsa staining, but sometimes they can be dis-
tinguished: Chromosome 3 is slightly larger, and chromosome 2 may display a
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