Repeated sequences in the DNA of Drosophila and their localization in giant chromosomes

It is shown by isopycnic density gradient centrifugation that the DNAs of the sibling species Drosophila hydei, Drosophila neohydei and Drosophila pseudoneohydei differ regarding the numbers and proportions of satellite DNA bands. An overwhelming proportion of all repetitive nucleotide sequences of the DNA is contained in these satellite fractions. The majority of the satellites are species specific despite the close phylogenetic and cytological relationship between the three species studied. — By in situ hybridization experiments it is demonstrated that the various satellite sequences occupy different positions within the chromosomes. All types of localization patterns, from a wide spread occurrence in all chromosomes to an apparent restriction to kinetochore regions of single chromosomes, have been observed. Main band DNA, on the other hand, in its hybridization behavior reflects the DNA distribution according to the banding pattern in giant chromosomes. Generally satellite sequences seem to be included in -heterochromatic chromosome regions but no relation to the heterochromatin of the Y-chromosome was found. — Renaturation studies support various evidence that satellite sequences occur in tandemly repetitious units. At least some of this repetitious material seems to be linked to non-satellite DNA sequences or to DNA of other satellites.     

Simple GA C T A repeats characterize the X chromosomal heterochromatin of Microtus agrestis,European field vole (Rodentia,Cricetidae)

The sex chromosomes of Microtus agrestis are extremely large due to the accumulation of constitutive heterochromatin. We have identified two prominent satellite bands of 2.0 and 2.8 kb in length after HaeIII and HinfI restriction enzyme digestion of genomic DNA, respectively. These satellites are located on the heterochromatic long arm of the X chromosome as shown using Microtus x mouse somatic cell hybrids. By in-gel hybridization with oligonucleotide probes, the organization of the two satellites was studied: among the many copies of the simple tandem tetranucleotide repeat GATA are interspersed rare single GACA tetramers. One of the satellites also harbours related GGAT simple tandem repeats. In situ hybridizations with plasmid-carried or oligonucleotide GA _C ^T A probes show clustered silver grains on the long and short arm of the X chromosome. Interspersion of differently organized (GATA)_n elements is also demonstrable in the autosomal complement and on the Y chromosome. These results are discussed in the context of the evolution of vertebrate sex chromosomes in relation to heterochromatin and simple repetitive DNA sequences.     

Chromosomal localization of human satellites 2 and 3 by a FISH method using oligonucleotides as probes

Classical satellites I, II and III are composed of a mixture of repeated sequences. However, each of them contains a simple family of repeated sequences as a major component. Satellites 2 and 3 are simple families of repeated sequences that form the bulk of human classical satellites II and III, respectively, and are composed of closely related sequences based on tandem repeats of the pentamer ATTCC. For this reason, extensive cross-hybridizations are probably responsible for the similar in situ hybridization patterns obtained for satellites II and III. We have used a fluorescent in situ hybridization method with highly specific oligonucleotides for satellites 2 and 3, respectively, as probes. Our results show that satellite 2 is mainly located on chromosomes 1, 2, 10 and 16, whereas the major domain of satellite 3 is on chromosome 9. Furthermore, minor sites of satellites 2 and 3 are shown. Two-colour in situ hybridizations have enabled us to define the spatial relationships existing between the major domains of both satellites and centromeric alpha satellite sequences. These experiments indicate that the heterochromatin regions of chromosomes 1, 9 and 16 have different molecular organizations.     

Towards a Drosophila genome map.

A physical map of the genome of Drosophila melanogaster has been created using 965 yeast artificial chromosome (YAC) clones assigned to locations in the cytogenetic map by in situ hybridization with the polytene salivary gland chromosomes. Clones with insert sizes averaging about 200 kb, totaling 1.7 genome equivalents, have been mapped. More than 80% of the euchromatic genome is included in the mapped clones, and 75% of the euchromatic genome is included in 161 cytological contigs ranging in size up to 2.5 Mb (average size 510 kb). On the other hand, YAC coverage of the one-third of the genome constituting the heterochromatin is incomplete, and clones containing long tracts of highly repetitive simple satellite DNA sequences have not been recovered.     

Studies of He-T DNA sequences in the pericentric regions of Drosophila chromosomes

He-T DNA is a complex set of repeated DNA sequences with sharply defined locations in the polytene chromosomes of Drosophila melanogaster. He-T sequences are found only in the chromocenter and in the terminal (telomere) band on each chromosome arm. Both of these regions appear to be heterochromatic and He-T sequences are never detected in the euchromatic arms of the chromosomes (Young et al. 1983). In the study reported here, in situ hybridization to metaphase chromosomes was used to study the association of He-T DNA with heterochromatic regions that are under-replicated in polytene chromosomes. Although the metaphase Y chromosome appears to be uniformly heterochromatic, He-T DNA hybridization is concentrated in the pericentric region of both normal and deleted Y chromosomes. He-T DNA hybridization is also concentrated in the pericentric regions of the autosomes. Much lower levels of He-T sequences were found in pericentric regions of normal X chromosomes; however compound X chromosomes, constructed by exchanges involving Y chromosomes, had large amounts of He-T DNA, presumably residual Y sequences. The apparent co-localization of He-T sequences with satellite DNAs in pericentric heterochromatin of metaphase chromosomes contrasts with the segregation of satellite DNA to alpha heterochromatin while He-T sequences hybridize to beta heterochromatin in polytene nuclei. This comparison suggests that satellite sequences do not exist as a single block within each chromosome but have interspersed regions of other sequences, including He-T DNA. If this is so, we assume that the satellite DNA blocks must associate during polytenization, leaving the interspersed sequences looped out to form beta heterochromatin. DNA from D. melanogaster has many restriction fragments with homology to He-T sequences. Some of these fragments are found only on the Y. Two of the repeated He-T family restriction fragments are found entirely on the short arm of the Y, predominantly in the pericentric region. Under conditions of moderate stringency, a subset of He-T DNA sequences cross-hybridizes with DNA from D. simulans and D. miranda. In each species, a large fraction of the cross-hybridizing sequences is on the Y chromosome.     

Karyotype and genome characterization in four cartilaginous fishes

Different approaches can be used to elucidate the unsolved questions concerning taxonomic evolution in cartilaginous fish. The study of the karyological characteristics of these vertebrates by combining molecular and traditional techniques of chromosome preparation and banding has been demonstrated to be a very effective method. In this paper we studied the localization and the composition of the constitutive heterochromatin by using C- and restriction endonuclease-banding in four selachian species, belonging to two of the four superorders. We also characterized two different types of repetitive genomic sequences in these species: satellite DNA and (TTAGGG)(n) telomeric sequences. Finally, we analysed the nuclear ribosomal gene to determine the number of the nucleolar organizers and their position on chromosomes by using silver staining, chromomycin A(3), and FISH (fluorescent in situ hybridization). The results showed a prevailingly telomeric localization of constitutive heterochromatin in the Galeomorphii, the presence of additional nucleolar organizer sites in Raja asterias, an exclusively telomeric localization of the (TTAGGG)(n) sequences in Scyliorhinus stellaris and both telomeric and interstitial in Taeniura lymma. These data, together with those concerning the conservation of the satellite DNA, seem to support the hypothesis that Chondrichthyes have an evolutionary history leading them to the acquisition of large genomes rich in highly repeated sequences and subjected to some selective pressures favoring the conservation of this DNA fraction.     

Integrated cytogenetic map of mitotic metaphase chromosome 9 of maize: resolution,sensitivity, and banding paint development

To study the correlation of the sequence positions on the physical DNA finger print contig (FPC) map and cytogenetic maps of pachytene and somatic maize chromosomes, sequences located along the chromosome 9 FPC map approximately every 10 Mb were selected to place on maize chromosomes using fluorescent in situ hybridization (FISH). The probes were produced as pooled polymerase chain reaction products based on sequences of genetic markers or repeat-free portions of mapped bacterial artificial chromosome (BAC) clones. Fifteen probes were visualized on chromosome 9. The cytological positions of most sequences correspond on the pachytene, somatic, and FPC maps except some probes at the pericentromeric regions. Because of unequal condensation of mitotic metaphase chromosomes, being lower at pericentromeric regions and higher in the arms, probe positions are displaced to the distal ends of both arms. The axial resolution of FISH on somatic chromosome 9 varied from 3.3 to 8.2 Mb, which is 12-30 times lower than on pachytene chromosomes. The probe collection can be used as chromosomal landmarks or as a "banding paint" for the physical mapping of sequences including transgenes and BAC clones and for studying chromosomal rearrangements.     

Satellite DNA of Anopheles stephensi liston (Diptera: Culicidae)

Four satellite DNAs in the Anopheles stephensi genome have been defined on the basis of their banding properties in Hoechst 33258-CsCl density gradients. Two of these satellites, satellites I and II, are visible on neutral CsCl density gradients as a light density peak forming approximately 15% of total cellular DNA. Hoechst-CsCl density gradient profiles of DNA extracted from polytene tissues indicates that these satellites are underreplicated in larval salivary gland cells and adult female Malpighian tubules and possibly also in ovarian nurse cells. The chromosomal location of satellite I on mitotic and polytene chromosomes has been determined by in situ hybridisation. Sequences complementary to satellite I are present in approximately equal amounts on a heterochromatic arm of the X and Y chromosomes and are also present, in smaller amounts, at the centromere of chromosome 3. A quantitative analysis of the in situ hybridisation experiments indicates that sequences complementary to satellite I at these two sites differ in their replicative behaviour during polytenisation: heterosomal satellite I sequences are under-replicated relative to chromosome 3 sequences in polytene larval salivary gland and ovarian nurse cell nuclei.     

Cytogenetic analysis of the third chromosome heterochromatin of Drosophila melanogaster

Previous cytological analysis of heterochromatic rearrangements has yielded significant insight into the location and genetic organization of genes mapping to the heterochromatin of chromosomes X, Y, and 2 of Drosophila melanogaster. These studies have greatly facilitated our understanding of the genetic organization of heterochromatic genes. In contrast, the 12 essential genes known to exist within the mitotic heterochromatin of chromosome 3 have remained only imprecisely mapped. As a further step toward establishing a complete map of the heterochomatic genetic functions in Drosophila, we have characterized several rearrangements of chromosome 3 by using banding techniques at the level of mitotic chromosome. Most of the rearrangement breakpoints were located in the dull fluorescent regions h49, h51, and h58, suggesting that these regions correspond to heterochromatic hotspots for rearrangements. We were able to construct a detailed cytogenetic map of chromosome 3 heterochromatin that includes all of the known vital genes. At least 7 genes of the left arm (from l(3)80Fd to l(3)80Fj) map to segment h49-h51, while the most distal genes (from l(3)80Fa to l(3)80Fc) lie within the h47-h49 portion. The two right arm essential genes, l(3)81Fa and l(3)81Fb, are both located within the distal h58 segment. Intriguingly, a major part of chromosome 3 heterochromatin was found to be "empty," in that it did not contain either known genes or known satellite DNAs.     

Cytological studies of heterochromatin function in the Drosophila melanogaster male: autosomal meiotic pairing

In Drosophila melanogaster it is now documented that the different satellite DNA sequences make up the majority of the centromeric heterochromatin of all chromosomes. The most popular hypothesis on this class of DNA is that satellite DNA itself is important to the pairing processes of chromosomes. Evidence in support of such a hypothesis is, however, circumstantial. This hypothesis has been evaluated by direct cytological examination of the meiotic behaviour of heterochromatically and/or euchromatically rearranged autosomes in the male. It was found that neither substantial deletions nor rearrangements of the autosomal heterochromatin cause any disruption of meiotic pairing. Autosomal pairing depends on homologs retaining sufficient euchromatic homology. This is the first clear demonstration that the highly repeated satellite DNA sequences in the heterochromatin of the second, third and fourth chromosomes are not important in meiotic pairing, but rather that some euchromatic homology in the autosomes is essential to ensure a regular meiotic process. These results on the autosomes, when taken in conjunction with our previous studies on sex chromosome pairing, clearly indicate that satellite DNA is not crucial for male meiotic chromosome pairing of any member of the D. melanogaster genome.     

Cytological dissection of sex chromosome heterochromatin of Drosophila hydei

Prophase chromosomes of Drosophila hydei were stained with 0.5 g/ml Hoechst 33258 and examined under a fluorescence microscope. While autosomal and X chromosome heterochromatin are homogeneously fluorescent, the entirely heterochromatic Y chromosome exhibits an extremely fine longitudinal differentiation, being subdivided into 18 different regions defined by the degree of fluorescence and the presence of constrictions. Thus high resolution Hoechst banding of prophase chromosomes provides a tool comparable to polytene chromosomes for the cytogenetic analysis of the Y chromosome of D. hydei. — D. hydei heterochromatin was further characterized by Hoechst staining of chromosomes exposed to 5-bromodeoxyuridine for one round of DNA replication. After this treatment the pericentromeric autosomal heterochromatin, the X heterochromatin and the Y chromosome exhibit numerous regions of lateral asymmetry. Moreover, while the heterochromatic short arms of the major autosomes show simple lateral asymmetry, the X and the Y heterochromatin exhibit complex patterns of contralateral asymmetry. These observations, coupled with the data on the molecular content of D. hydei heterochromatin, give some insight into the chromosomal organization of highly and moderately repetitive heterochromatic DNA.     

Y chromosomal DNA of Drosophila hydei

Six recombinant DNA clones are described, which are derived from the Y chromosome of Drosophila hydei. They reveal characteristic features of Y chromosomal DNA sequences. Three of the cloned inserts are Y-specific and are members of the same family of repeated sequences associated with the lampbrush loop-forming fertility gene "nooses" in the short arm of the Y chromosome. The other three cloned sequences are members of three different families of repeated sequences, but display a small amount of homology to one another and to the family of the nooses sequences. These three cloned sequences are found preferentially in the Y chromosome, but also in other chromosomal positions. The Y chromosomal copies are located in the short arm of the Y chromosome. The other copies are found in autosomal kinetochore-associated heterochromatin or, for one of the cloned sequences, in one band of the giant chromosome 4, in addition to the kinetochore heterochromatin.     

The Drosophila Salivary Gland Chromocenter Contains Highly Polytenized Subdomains of Mitotic Heterochromatin

Peri-centromeric regions of Drosophila melanogaster chromosomes appear heterochromatic in mitotic cells and become greatly underrepresented in giant polytene chromosomes, where they aggregate into a central mass called the chromocenter. We used P elements inserted at sites dispersed throughout much of the mitotic heterochromatin to analyze the fate of 31 individual sites during polytenization. Analysis of DNA sequences flanking many of these elements revealed that middle repetitive or unique sequence DNAs frequently are interspersed with satellite DNAs in mitotic heterochromatin. All nine Y chromosome sites tested were underrepresented >20-fold on Southern blots of polytene DNA and were rarely or never detected by in situ hybridization to salivary gland chromosomes. In contrast, nine tested insertions in autosomal centromeric heterochromatin were represented fully in salivary gland DNA, despite the fact that at least six were located proximal to known blocks of satellite DNA. The inserted sequences formed diverse, site-specific morphologies in the chromocenter of salivary gland chromosomes, suggesting that domains dispersed at multiple sites in the centromeric heterochromatin of mitotic chromosomes contribute to polytene β-heterochromatin. We suggest that regions containing heterochromatic genes are organized into dispersed chromatin configurations that are important for their function in vivo.     

The biological significance of variation in satellite DNA and heterochromatin in newts of the genus Triturus: an evolutionary perspective

The functional and evolutionary significance of highly repetitive, simple sequence (satellite) DNA is analysed by examining available information on the patterns of variation of heterochromatin and cloned satellites among newts (family Salamandridae), and particularly species of the European genus Triturus. This information is used to develop a model linking evolutionary changes in satellite DNAs and chromosome structure. In this model, satellites accumulate initially in large tandem blocks around centromeres of some or all of the chromosomes, mainly by repeated chromosomal exchanges in these regions. Centromeric blocks later become broken up and dispersed by small, random chromosome rearrangements in these regions. They are dispersed first to pericentric locations and then gradually more distally into the chromosome arms and telomeres. Dispersal of a particular satellite is accompanied by changes in sequence structure (for example, base substitutions, deletions, etc.) and a corresponding decrease in its detectability at either the molecular or cytological level. On the basis of this model, observed satellites in newt species may be classified as 'old', 'young', or of 'intermediate' phylogenetic age. The functions and effects of satellite DNA and heterochromatin at the cellular and organismal levels are also discussed. It is suggested that satellite DNA may have an impact on cell proliferation through the effect of late-replicating satellite-rich heterochromatin on the duration of S-phase of the cell cycle. It is argued that even small alterations in cell cycle time due to changes in heterochromatin amount may have magnified effects on organismal growth that may be of adaptive significance.     

The location of highly repetitious DNA in the somatic chromosomes of Drosophila melanogaster

In situ hybridization of Drosophila melanogaster somatic chromosomes has been used to demonstrate the near exact correspondence between the location of highly repetitious DNA and classically defined constitutive heterochromatin. The Y chromosome, in particular, is heavily labeled even by cRNA transcribed from female (XX) DNA templates (i.e., DNA from female Drosophila with 2 Xs and 2 sets of autosomes). This observation confirms earlier reports that the Y chromosome contains repeated DNA sequences that are shared by other chromosomes. In grain counting experiments the Y chromosome shows significantly heavier label than any other chromosome when hybridized with cRNA from XY DNA templates (i.e., DNA from male Drosophila with 1 X and 1 Y plus 2 sets of autosomes). However, the preferential labeling of the Y is abolished if the cRNA is derived from XX DNA. We interpret these results as indicating the presence of a class of Y chromosome specific repeated DNA in D. melanogaster. The relative inefficiency of the X chromosome in binding cRNA from XY and XYY DNA templates, coupled with its ability to bind XX derived cRNA, may also indicate the presence of an X chromosome specific repeated DNA.     

Homologies between X and Y chromosomes detected by DNA probes: localisation and evolution.

We have isolated and characterized DNA probes that detect homologies between the X and Y chromosomes. Clone St25 is derived from the q13-q22 region of the X chromosome and recognizes a 98% homologous sequence on the Y chromosome. Y specific fragments were present in DNAs from 5 Yq-individuals and from 4 out of 7 XX males analysed. An X linked TaqI RFLP is detected with the St25 probe (33% heterozygosity) which should allow one to establish a linkage map including other polymorphic X-Y homologous sequences in this region and to compare it to a Y chromosome deletion map. Probe DXS31 located in Xp223-pter detects a 80% homologous sequence in the Y chromosome. The latter can be assigned to Yq11-qter outside the region which contains the Y specific satellite sequences. ACT1 and ACT2, the actin sequences present on the X and Y chromosomes respectively, have been cloned. No homology was detected between the X and Y derived fragments outside from the actin sequence. ACT2 and the Y specific sequence corresponding to DXS31 segregate together in a panel of Y chromosomes aberrations, and might be useful markers for the region important for spermatogenesis in Yq. Various primate species were analysed for the presence of sequences homologous to the three probes. Sequences detected by St25 and DXS31 are found only on the X chromosome in cercopithecoidae. The sequences which flank ACT2 detect in the same species autosomal fragments but no male specific fragments. It is suggested that the Y chromosome acquired genetic material from the X chromosome and from autosomes at various times during primate evolution.     

A novel simple satellite DNA is colocalized with the Stalker retrotransposon in Drosophila melanogaster heterochromatin

In the T(1;2)dor ^var7 multibreak rearrangement the distal 1A-2B segment of the X chromosome of Drosophila melanogaster is juxtaposed to an inverted portion of the heterochromatin of chromosome 2. Analysis of mitotic chromosomes by a series of banding techniques has permitted us precisely to locate the heterochromatic breakpoint of this translocation in the h42 region of 2R. Cloning and sequencing of the eu-heterochromatic junction revealed that the translocated 1A-2B fragment is joined to (AACAC)_n repeats, which represent a previously undescribed satellite DNA in D. melanogaster. These repeated sequences have been estimated to account for about 1 Mb of the D. melanogaster genome. The repeats are located mainly in the Y chromosome and in the heterochromatin of the right arm of chromosome 2 (2Rh), where they are colocalized with the Stalker retrotransposon. Received: 3 October 1998 / Accepted: 3 December 1998     

An alpha satellite DNA polymorphism specific for the centromeric region of chromosome 13

Alpha satellite DNA is composed of variants of a short consensus sequence that are repeated in tandem arrays in the centromeric heterochromatin of each human chromosome. To define centromeric markers for linkage studies, we screened human genomic DNA for restriction fragment length polymorphisms using a probe detecting alphoid sequences on chromosomes 13 and 21. We describe one such DNA polymorphism. Analysis of linkage of this DNA marker to other polymorphic markers in the CEPH pedigrees demonstrates linkage to markers on the proximal long arm of chromosome 13 and defines the centromeric end of the linkage map of this chromosome.     

A physical map of the polytenized region (101EF-102F) of chromosome 4 in Drosophila melanogaster

Chromosome 4, the smallest autosome ( approximately 5 Mb in length) in Drosophila melanogaster contains two major regions. The centromeric domain ( approximately 4 Mb) is heterochromatic and consists primarily of short, satellite repeats. The remaining approximately 1.2 Mb, which constitutes the banded region (101E-102F) on salivary gland polytene chromosomes and contains the identified genes, is the region mapped in this study. Chromosome walking was hindered by the abundance of moderately repeated sequences dispersed along the chromosome, so we used many entry points to recover overlapping cosmid and BAC clones. In situ hybridization of probes from the two ends of the map to polytene chromosomes confirmed that the cloned region had spanned the 101E-102F interval. Our BAC clones comprised three contigs; one gap was positioned distally in 102EF and the other was located proximally at 102B. Twenty-three genes, representing about half of our revised estimate of the total number of genes on chromosome 4, were positioned on the BAC contigs. A minimal tiling set of the clones we have mapped will facilitate both the assembly of the DNA sequence of the chromosome and a functional analysis of its genes.