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 2E9ABSTRACT

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.

AcknowledgementsWe 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.

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.

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.

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 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 andvir = D. virilis. Sizes are the locations determined from a 1 kb marker ladder (GIBCO-BRL).

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 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.