The heterochromatic properties and physical organization of chromosome 4 in Drosophila

1
The heterochromatic properties and physical
organization of chromosome 4 in Drosophila R.
Hodgetts, L. Podemski, N. Aippersbach, L. Howard
and J. Locke Department of Biological Sciences,
University of Alberta, Edmonton, Alberta, Canada,
T6G 2E9
ABSTRACT Genetic analysis of Drosophila
melanogaster chromosome 4 has been hindered by
its lack of crossing over and the abundance of
repeated sequences. The unusual properties also
include a diffuse banded appearance in polytene
chromosomes and the variegation of P element
transgenes. To facilitate our investigation of
this small chromosome, we are constructing a
physical map of the banded portion. Our initial
efforts focused on assembling cosmid clones into
contigs, but several regions were not represented
in our libraries which. We have recently begun
screening a new genomic library constructed using
a Bacterial Artificial Chromosome (BAC) vector.
Many of the gaps in our cosmid contig map have
now been filled using these BAC clones. Our
progress in positioning known genes on the map
will be presented. As part of this mapping
project we sequenced two cosmid clones,
representing 5 of the euchromatic region. Both
clones contained numerous short repeated DNA
sequences as identified by cross hybridization
with labeled genomic DNA. One such sequence,
DINE-1 (for Drosophila INterspersed Element -1),
which bears a superficial resemblance to
mammalian SINEs, is present roughly every 3.5 kb.
A comparison of individual DINE-1 elements
revealed that there are two conserved domains
separated by a variable spacer. DINE-1 displays
an unusual genomic distribution biased primarily
toward chromosome 4 and heterochromatic regions
in the genomes of the two sibling species D.
melanogaster and D. simulans, whose dot
chromosomes exhibit poorly resolved polytene
bands. Interestingly, the element is absent from
the dot chromosome of D. virilis, which exhibits
a well defined banded structure. These
observations suggest that DINE-1 contributes to
the heterochromatic nature of chromosome 4 in D.
melanogaster.
An in situ hybridization to localize the BAC
clones in the adjacent chromosome 4 contig. The
proximal and distal BAC clones were probed to a
T(14)wm5 chromosome and detected with rhodamine
(red) against DAPI (blue) stained chromosomes.
The proximal BAC is located in the most proximal
region while the distal BAC is located at the
telomere of chromosome 4.
Progress on physical map of chromosome 4.
Chromosome 4 consists of cytogenetic regions
101EF to 102F. The locations of mapped loci and
BAC and cosmid clones are shown relative to each
other. BAC clones are from the BACN and BAC
libraries of the CEPH and UK HGMP Resource Centre
and the RPCI-98 BAC library of the Drosophila
Genome Project. Cosmid clones are primarily from
our own libraries. Chromosome 4 loci are listed
above the clones.
DINE-1 a short repeated element with two
conserved domains. Dot matrix analysis
comparisons of the archetypal DINE-1 element 1F.
All comparisons are shown at the same scale
using the DNA Strider computer program set to
require matches of 11 of 15 bp for a dot in the
matrix. Large arrows indicate the locations of
the repeat within the 1F element. Conserved
domains A and B are label in each plot. a)- A
schematic of the DINE-1 1F element. The
organization of the conserved blocks A and B
(solid regions) and the variable spacer region
(open region) is shown. The numbered arrows
depict the locations and orientations of the four
PCR primers used to amplify the DINE-1 as
described in the Materials and Methods. b)- The
DINE-1 1F element compared to sequences 2900-3500
adjacent to the msl-2 locus.c)- The DINE-1 1F
element compared to sequences 3900-4400 adjacent
to the 68C glue protein gene.d)- The DINE-1 1F
element compared to sequences 1500-2100 adjacent
to the U1-82.1 gene.e)- The 2900-3500 adjacent
to the msl-2 locus compared to sequences
3900-4400 adjacent to the 68C glue protein
gene.f)- The 2900-3500 adjacent to the msl-2
locus compared to sequences 1500-2100 adjacent to
the U1-82.1 gene.g)- The DINE-1 1F element
compared to its antiparallel equivalent showing
no inverted repeats.
DINE-1 is not present in many other insects
species. Autoradiograms of genomic Southern
transfers probed with the entire DINE-1 T probe
is radiolabelled PCR product, generated by
primers 1 and 4 (Fig. 1a). Genomic DNAs from
humans, Tenebrio molitor, Apis melifora, Cotesia
congregata, Manduca sexta, Glossina marsitans,
Sarcophaga bullata, Musca domestica and
Drosophila melanogaster was digested with Alu I
and treated as in part a).
DINE-1 is localized to chromosome
4. Autoradiograms of Southern transfers showing
hybridization of the entire DINE-1 element to
sets of cosmid DNAs. Cosmid DNA was probed with
radiolabelled PCR product generated with primers
1 and 4 using the cosL7L template (Fig. 1).
Washes were conducted at low stringency. a)
Lanes 1-18 various cosmids localized to
chromosome 4. b) Lanes 2-14 cosmids localized
to chromosomes other than 4. The positive control
in lanes 1 and 15 is cosL7L DNA .
DINE-1 is found in heterochromatin. In situ
localization of DINE-1 on salivary gland polytene
chromosomes. The DINE-1 PCR product obtained by
amplification of the cosL7L template using
primers 1 and 4 (Fig. 1) was labeled with
digoxigenin-11-dUTP and hybridized to salivary
gland chromosomes. The locations of chromosome 4
in melanogaster and simulans and its homologue in
virilis, chromosome 6, are identified with black
arrows.Parts a and b are high and low,
respectively, concentration probings of D.
melanogaster strain T(14)wm5, in which
chromosome 4 has been translocated to the end of
the X chromosome. Parts c and d are high and low,
respectively, probings of wild type D. simulans.
Parts e and f are high and low, respectively,
probings of wild type D. virilis. Part g is an
enlargement of the staining of D. melanogaster
chromosome 4 at the tip of the T(14)wm5
translocation. The white arrow indicates the
break point with stained chromosome 4 on the
right and part of the unstained X chromosome on
the left.
Autoradiograms of Southern transfers showing a
comparison of the DINE-1 block A and block B
hybridizations to a set of 18 cosmids localized
to chromosome 4. Hybridizations were conducted
at at low stringency. a). The transfer shown in
Fig. 3a was stripped and re-probed with the A
block probe (a radiolabeled 293 bp PCR fragment
generated using primers 1 and 2 Fig. 1a).b).
Radioactivity was stripped from the membrane in
a) and the blot was re-hybridized to a B block
probe (99 bp radiolabeled PCR fragment generated
using primers 3 and 4 Fig. 1).
DINE-1 is present in other Drosophila
species. Autoradiograms of Southern transfers of
Drosophila genomic DNA probed with the entire
DINE-1 The probe is radiolabelled PCR product
generated by primers 1 and 4 (Fig. 1a). a).
Genomic DNAs from three strains of Drosophila
melanogaster were digested with Alu I, Sau3A I,
EcoR I and Hind III, separated and a Southern
blot carried out. The membrane was hybridized at
65C and washed at low stringency. mel D.
melanogaster, sim D. simulans and vir D.
virilis. Sizes are the locations determined from
a 1 kb marker ladder (GIBCO-BRL).
Acknowledgements We thank Effie Woloshyn for
assistance with an in situ hybridization and Dr.
A. Keddie for supplying some of the insect
species. We also thank the many researchers
who gave their chromosome 4 sequence probes that
we have used to identify the P1, cosmid, and BAC
clones in this work J.L. Couderc, R.W. Levis,
S. Kunes, C. Sung, S.N. Robinow, P.Callaerts,
M.-l. A. Joiner, B. Stronach, E. Frei, P. Lasko,
A. Worthington, S. Russel, J.A. Kassis, S.C.R.
Elgin, L.L. Wallrath, R. K. Kutty, M.Winberg,
C.S. Goodman, Y. Grau, E.A. Fyrberg, J.N.
Noordermeer, C.J. O'Kane, S. Sweeney, K. Arora,
T.E. Haerry, R. Dubreuil, A.R. Campos and others.
This work was funded by the Medical Research
Council of Canada and the Natural Sciences and
Engineering Research Council of Canada.