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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(1;4)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 .
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 65°C 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 (GIBCOBRL).
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(1;4)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(1;4)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.
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.