Structure of DNA near long tandem arrays of alpha satellite DNA at the centromere of human chromosome 7.

The centromeric regions of human chromosomes contain long tracts of tandemly repeated DNA, of which the most extensively characterized is alpha satellite. In a screen for additional centromeric DNA sequences, four phage clones were obtained which contain alpha satellite as well as other sequences not usually found associated with tandemly repeated alpha satellite DNA, including L1 repetitive elements, an Alu element, and a novel AT-rich repeated sequence. The alpha satellite DNA contained within these clones does not demonstrate the higher-order repeat structure typical of tandemly repeated alpha satellite. Two of the clones contain inversions; instead of the usual head-to-tail arrangement of alpha satellite monomers, the direction of the monomers changes partway through each clone. The presence of both inversions was confirmed in human genomic DNA by polymerase chain reaction amplification of the inverted regions. One phage clone contains a junction between alpha satellite DNA and a novel low-copy repeated sequence. The junction between the two types of DNA is abrupt and the junction sequence is characterized by the presence of runs of A's and T's, yielding an overall base composition of 65% AT with local areas > 80% AT. The AT-rich sequence is found in multiple copies on chromosome 7 and homologous sequences are found in (peri)centromeric locations on other human chromosomes, including chromosomes 1, 2, and 16. As such, the AT-rich sequence adjacent to alpha satellite DNA provides a tool for the further study of the DNA from this region of the chromosome. The phage clones examined are located within the same 3.3-Mb SstII restriction fragment on chromosome 7 as the two previously described alpha satellite arrays, D7Z1 and D7Z2. These new clones demonstrate that centromeric repetitive DNA, at least on chromosome 7, may be more heterogeneous in composition and organization than had previously been thought.     

Replication of centromere II of Schizosaccharomyces pombe.

The centromeric DNAs of Schizosaccharomyces pombe chromosomes resemble those of higher eukaryotes in being large and composed predominantly of repeated sequences. To begin a detailed analysis of the mode of replication of a complex centromere, we examined whether any sequences within S. pombe centromere II (cen2) have the ability to mediate autonomous replication. We found a high density of segments with such activity, including at least eight different regions comprising most of the repeated and unique centromeric DNA elements. A physical mapping analysis using two-dimensional gels showed that autonomous replication initiated within the S. pombe sequences in each plasmid. A two-dimensional gel analysis of replication on the chromosomes revealed that the K and L repeat elements, which occur in multiple copies at all three centromeres and comprise approximately 70% of total centromeric DNA mass in S. pombe, are both sites of replication initiation. In contrast, the unique cen2 central core, which contains multiple segments that can support autonomous replication, appears to be repressed for initiation on the chromosome. We discuss the implications of these findings for our understanding of DNA replication and centromere function.     

Location of repetitious DNA in the chromosomes of the desert locust (Schistocerca gregaria)

A modified Giemsa staining technique and the in situ hybridisation technique, have been used to investigate the localisation of highly repeated sequences in the karyotype of the locust Schistocerca gregaria. The centromeric regions are stained densely with Giemsa and further Giemsa-stained bands occur at the telomeric region of the short (S) chromosomes. RNA complementary to repetitious DNA hybridised to loci scattered along the whole length of the chromosomes, with concentrations of grains at the centromeric regions of all the chromosomes and also at the telomeric regions of the S chromosomes.     

Restrictive localization of centromeric heterochromatin (C-bands) in Presbytis e. entellus (Dufresne) as compared to Macaca mulatta (Zimmerman)

C-bands are observed in the centromeric regions of only three pairs of autosomes and the distal portion of the small acrocentric Y in a total complement of 44 chromosomes of a male Presbytis e. entellus. Simultaneously treated slides of a Rhesus monkey, however, have C-bands in all the 42 chromosomes. The lack of C-bands may be due to (1) absence of highly repetitive DNA in the centromeric region of certain chromosomes or (2) presence of minute quantity of such DNA which is imperceptible or (3) different types of centromeric heterochromatin with a varying degree of repetition of DNA sequences all of which do not react in similar manner to various techniques employed at present. It is hypothesized that the centromeric heterochromatin rich in satellite DNA helps in withstanding the force of excessive coiling of chromosomes at the centromere to facilitate the functioning of the genes for microtubular protein during cell division when other genes are rendered inactive due to compactness of chromosomes.     

Novel sequencing strategy for repetitive DNA in a Drosophila BAC clone reveals that the centromeric region of the Y chromosome evolved from a telomere

The centromeric and telomeric heterochromatin of eukaryotic chromosomes is mainly composed of middle-repetitive elements, such as transposable elements and tandemly repeated DNA sequences. Because of this repetitive nature, Whole Genome Shotgun Projects have failed in sequencing these regions. We describe a novel kind of transposon-based approach for sequencing highly repetitive DNA sequences in BAC clones. The key to this strategy relies on physical mapping the precise position of the transposon insertion, which enables the correct assembly of the repeated DNA. We have applied this strategy to a clone from the centromeric region of the Y chromosome of Drosophila melanogaster. The analysis of the complete sequence of this clone has allowed us to prove that this centromeric region evolved from a telomere, possibly after a pericentric inversion of an ancestral telocentric chromosome. Our results confirm that the use of transposon-mediated sequencing, including positional mapping information, improves current finishing strategies. The strategy we describe could be a universal approach to resolving the heterochromatic regions of eukaryotic genomes.     

Study of alpha-satellite DNA in cosmid libraries, specific for chromosomes 13, 21, and 22, using fluorescence in situ hybridization

Fluorescent in situ hybridization (FISH) was employed in mapping the alpha-satellite DNA that was revealed in the cosmid libraries specific for human chromosomes 13, 21, and 22. In total, 131 clones were revealed. They contained various elements of centromeric alphoid DNA sequences of acrocentric chromosomes, including those located close to SINEs, LINEs, and classical satellite sequences. The heterochromatin of acrocentric chromosomes was shown to contain two different groups of alphoid sequences: (1) those immediately adjacent to the centromeric regions (alpha 13-1, alpha 21-1, and alpha 22-1 loci) and (2) those located in the short arm of acrocentric chromosomes (alpha 13-2, alpha 21-2, and alpha 22-2 loci). Alphoid DNA sequences from the alpha 13-2, alpha 21-2, and alpha 22-2 loci are apparently not involved in the formation of centromeres and are absent from mitotically stable marker chromosomes with a deleted short arm. Robertsonian translocations t(13q; 21q) and t(14q; 22q), and chromosome 21p-. The heterochromatic regions of chromosomes 13, 21, and 22 were also shown to contain relatively chromosome-specific repetitive sequences of various alphoid DNA families, whose numerous copies occur in other chromosomes. Pools of centromeric alphoid cosmids can be of use in further studies of the structural and functional properties of heterochromatic DNA and the identification of centromeric sequences. Moreover, these clones can be employed in high-resolution mapping and in sequencing the heterochromatic regions of the human genome. The detailed FISH analysis of numerous alphoid cosmid clones allowed the identification of several new, highly specific DNA probes of molecular cytogenetic studies--in particular, the interphase and metaphase analyses of chromosomes 2, 9, 11, 14, 15, 16, 18, 20, 21-13, 22-14, and X.     

Cross chromosomal similarity for DNA sequence compression

Current DNA compression algorithms work by finding similar repeated regions within the DNA sequence and then encoding these regions together to achieve compression. Our study on chromosome sequence similarity reveals that the length of similar repeated regions within one chromosome is about 4.5% of the total sequence length. The compression gain is often not high because of these short lengths. It is well known that similarity exist among different regions of chromosome sequences. This implies that similar repeated sequences are found among different regions of chromosome sequences. Here, we study cross-chromosomal similarity for DNA sequence compression. The length and location of similar repeated regions among the sixteen chromosomes of S. cerevisiae are studied. It is found that the average percentage of similar subsequences found between two chromosome sequences is about 10% in which 8% comes from cross-chromosomal prediction and 2% from self-chromosomal prediction. The percentage of similar subsquences is about 18% in which only 1.2% comes from self-chromosomal prediction while the rest is from cross-chromosomal prediction among the 16 chromosomes studied. This suggests the importance of cross-chromosomal similarities in addition to self-chromosomal similarities in DNA sequence compression. An additional 23% of storage space could be reduced on average using self-chromosomal and cross-chromosomal predictions in compressing the 16 chromosomes of S. cerevisiae.     

Characterization of extrachromosomal DNA in the flesh fly Sarcophaga bullata

The polytene pupal foot pad cells of the flesh fly Sarcophaga bullata contain numerous extrachromosomal DNA containing granules. We have determined both the origin and the nature of the DNA sequences present in these granules. Studies done with quinacrine staining of seven day old pupal foot-pad polytene nuclei showed that the granules fluoresced very brightly while the chromosomal bands to which the granules were attached did not. The only other highly fluroescent regions of the polytene karyotype were the centromeric heterochromatin of chromosomes C and E and several bands associated with the nucleolus of Chromosome A. When polytene nuclei were hybridized in situ with cRNA made from highly repetitive DNA, many of the granules positively labeled. Most of the label on these slides was concentrated on the centromeric heterochromatin of chromosomes C and E. Quinacrine staining of the foot-pad cells at very early stages of pupal development showed that when granules were present, they were always closely associated with the same two centromeric regions, those of chromosomes C and E. Since the highly repetitive DNA located in these centromeric regions is underreplicated, we conclude that the granules result from an extrusion process which takes place early during the polytenization of these cells. The chromosomal integrity of the centromeric heterochromatin of chromosomes C and E is apparently disrupted and repetitive sequences are dissociated from the chromosomes as DNA granules which then secondarily become associated with chromosomal bands throughout the nucleus.     

Chromosome ends: different sequences may provide conserved functions

The structures of specific chromosome regions, centromeres and telomeres, present a number of puzzles. As functions performed by these regions are ubiquitous and essential, their DNA, proteins and chromatin structure are expected to be conserved. Recent studies of centromeric DNA from human, Drosophila and plant species have demonstrated that a hidden universal centromere-specific sequence is highly unlikely. The DNA of telomeres is more conserved consisting of a tandemly repeated 6-8 bp Arabidopsis-like sequence in a majority of organisms as diverse as protozoan, fungi, mammals and plants. However, there are alternatives to short DNA repeats at the ends of chromosomes and for telomere elongation by telomerase. Here we focus on the similarities and diversity that exist among the structural elements, DNA sequences and proteins, that make up terminal domains (telomeres and subtelomeres), and how organisms use these in different ways to fulfil the functions of end-replication and end-protection.     

Sequence and evolution of rhesus monkey alphoid DNA

Summary Analysis of rhesus monkey alphoid DNA suggests that it arose by tandem duplication of an ancestral monomer unit followed by independent variation within two adjacent monomers (one becoming more divergent than the other) before their amplification as a dimer unit to produce tandem arrays. The rhesus monkey alphoid DNA is a tandemly repeated, 343-bp dimer; the consensus dimer is over 98% homologous to the alphoid dimers reported for baboon and bonnet monkey, 81% homologous to the African green monkey alpha monomer, and less than 70% homologous to the more divergent human alphoid DNAs. The consensus dimer consists of two wings (I and II, 172 and 171 bp, respectively) that are only 70% homologous to each other, but share seven regions of exact homology. These same regions are highly conserved among the consensus sequences of the other cercopithecid alphoid DNAs. The three alpha-protein binding sites reported for African green monkey alpha DNA by F. Strauss and A. Varshavsky (Cell 37: 889–901, 1984) occur in wings I and II, but with one site altered in wing I. Two cloned dimer segments are 98% homologous to the consensus, each containing 8 single-base-pair differences within the 343-bp segment. Surprisingly, 37% of these differences occur in regions that are evolutionarily conserved in the alphoid consensus sequences, including the alpha-protein binding sites. Sequence variation in this highly repetitive DNA family may produce unique nucleosomal architectures for different members of an alphoid array. These unique architectures may modulate the evolution of these repetitive DNAs and may produce unique centromeric characteristics in primate chromosomes.     

Human (Homo sapiens) and chimpanzee (Pan troglodytes) share similar ancestral centromeric alpha satellite DNA sequences but other fractions of heterochromatin differ considerably

The euchromatic regions of chimpanzee (Pan troglodytes) genome share approximately 98% sequence similarity with the human (Homo sapiens), while the heterochromatic regions display considerable divergence. Positive heterochromatic regions revealed by the CBG-technique are confined to pericentromeric areas in humans, while in chimpanzees, these regions are pericentromeric, telomeric, and intercalary. When human chromosomes are digested with restriction endonuclease AluI and stained by Giemsa (AluI/Giemsa), positive heterochromatin is detected only in the pericentromeric regions, while in chimpanzee, telomeric, pericentromeric, and in some chromosomes both telomeric and centromeric, regions are positive. The DA/DAPI technique further revealed extensive cytochemical heterogeneity of heterochromatin in both species. Nevertheless, the fluorescence in situ hybridization technique (FISH) using a centromeric alpha satellite cocktail probe revealed that both primates share similar pericentromeric alpha satellite DNA sequences. Furthermore, cross-hybridization experiments using chromosomes of gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) suggest that the alphoid repeats of human and great apes are highly conserved, implying that these repeat families were present in their common ancestor. Nevertheless, the orangutan's chromosome 9 did not cross-hybridize with human probe. The euchromatic regions of chimpanzee (Pan troglodytes) genome share approximately 98% sequence similarity with the human (Homo sapiens), while the heterochromatic regions display considerable divergence. Positive heterochromatic regions revealed by the CBG-technique are confined to pericentromeric areas in humans, while in chimpanzees, these regions are pericentromeric, telomeric, and intercalary. When human chromosomes are digested with restriction endonuclease AluI and stained by Giemsa (AluI/Giemsa), positive heterochromatin is detected only in the pericentromeric regions, while in chimpanzee, telomeric, pericentromeric, and in some chromosomes both telomeric and centromeric, regions are positive. The DA/DAPI technique further revealed extensive cytochemical heterogeneity of heterochromatin in both species. Nevertheless, the fluorescence in situ hybridization technique (FISH) using a centromeric alpha satellite cocktail probe revealed that both primates share similar pericentromeric alpha satellite DNA sequences. Furthermore, cross-hybridization experiments using chromosomes of gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) suggest that the alphoid repeats of human and great apes are highly conserved, implying that these repeat families were present in their common ancestor. Nevertheless, the orangutan's chromosome 9 did not cross-hybridize with human probe. © 1995 Wiley-Liss, Inc.     

Molecular cytogenetics of the equidae. II. purification and cytological localization of a (G + C)-rich satellite DNA from Equus hemionus onager and cross-species hybridization to E. asinus chromosomes

A (G + C)-rich satellite DNA component (p = 1.716 g/ml) has been fractionated from the total DNA of the Iranian subspecies of the Asiatic wild ass, Equus hemionus onager, by successive dactinomycin-CsCl and netropsin sulfate-CsCl isopycnic gradients. Complementary 3H-RNA (cRNA) transcribed from the satellite DNA hybridized predominantly to the centromeric and telomeric constitutive heterochromatic regions of onager chromosomes. These studies have suggested that satellite DNA's with similar sequences are present in the centromeric, as well as telomeric, heterochromatic regions of some onager chromosomes. The centromeric region of the fusion metacentric t(23;24) of the onager is deficient in sequences homologous to the onager 1.716 g/ml satellite DNA, indicating a loss of satellite DNA during fusion or an amplification of the satellite DNA in the centromeric regions of the acrocentric chromosomes 23 and 24 subsequent to fission. Sequences complementary to onager 1.716 g/ml satellite DNA show extensive hybridization to the constitutive heterochromatin of the feral donkey (E. asinus) karyotype, consistent with a view of conservation and amplification of similar or identical sequences in the two species.     

A molecular and cytogenetic survey of major repeated DNA sequences in tomato (Lycopersicon esculentum)

Summary The major families of repeated DNA sequences in the genome of tomato (Lycopersicon esculentum) were isolated from a sheared DNA library. One thousand clones, representing one million base pairs, or 0.15% of the genome, were surveyed for repeated DNA sequences by hybridization to total nuclear DNA. Four major repeat classes were identified and characterized with respect to copy number, chromosomal localization by in situ hybridization, and evolution in the family Solanaceae. The most highly repeated sequence, with approximately 77000 copies, consists of a 162 bp tandemly repeated satellite DNA. This repeat is clustered at or near the telomeres of most chromosomes and also at the centromeres and interstitial sites of a few chromosomes. Another family of tandemly repeated sequences consists of the genes coding for the 45 S ribosomal RNA. The 9.1 kb repeating unit in L. esculentum was estimated to be present in approximately 2300 copies. The single locus, previously mapped using restriction fragment length polymorphisms, was shown by in situ hybridization as a very intense signal at the end of chromosome 2. The third family of repeated sequences was interspersed throughout nearly all chromosomes with an average of 133 kb between elements. The total copy number in the genome is approximately 4200. The fourth class consists of another interspersed repeat showing clustering at or near the centromeres in several chromosomes. This repeat had a copy number of approximately 2100. Sequences homologous to the 45 S ribosomal DNA showed cross-hybridization to DNA from all solanaceous species examined including potato, Datura, Petunia, tobacco and pepper. In contrast, with the exception of one class of interspersed repeats which is present in potato, all other repetitive sequences appear to be limited to the crossing-range of tomato. These results, along with those from a companion paper (Zamir and Tanksley 1988), indicate that tomato possesses few highly repetitive DNA sequences and those that do exist are evolving at a rate higher than most other genomic sequences.     

Genome plasticity in Festuca arundinacea: direct response to temperature changes by redundancy modulation of interspersed DNA repeats

The response of the genome of Festuca arundinacea seedlings to changes in the temperature at which they were grown was investigated. Fifteen repeated sequences in the nuclear DNA were isolated and hybridized to the genomic DNA of seedlings grown at 10 degrees C or 30 degrees C. The redundancies of sequences recognized by four probes ( FaA5, FaH8, FaH13 and FaH14), were found to differ significantly in the two DNAs. DNA sequences recognized by FaH8, FaH13 and FaH14 were more represented in the genome of the 30 degrees C-raised seedlings than in the genome of the 10 degrees C-raised seedlings (76.5 x 10(3), 1.9 x 10(3), and 111.8 x 10(3) copies per haploid, 1C genome vs 62.7 x 10(3), 1.3 x 10(3), and 80.8 x 10(3) copies, respectively). In contrast, FaA5-related sequences were more represented in the genome of seedlings grown at the lower temperature (15.5 x 10(3) vs 10.2 x 10(3) copies, respectively). Southern-blot hybridization of these repeats to digested genomic DNA produced patterns which indicated that the probe sequences were part of longer repeated sequences having a limited degree of structural heterogeneity. These patterns were partly different when the probes were hybridized to the DNA from seedlings grown at 10 degrees C or 30 degrees C. In situ hybridization showed that the DNA sequences recognized by each probe were scattered along the length of all the chromosomes, with preferential location of FaA5- and FaH13-related sequences at given, mainly centromeric, regions of certain chromosomes. These findings suggest that redundancy modulations of interspersed repeated sequences allow direct responses of the genome of F. arundinacea to changes in environmental temperature.     

Complex structure of knobs and centromeric regions in maize chromosomes

The recovery of maize (Zea mays L.) chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses enables us to analyze the structure and composition of individual maize chromosomes via the isolation and characterization of chromosome-specific cosmid clones. Restriction fragment fingerprinting, sequencing, and in situ hybridization were applied to discover a new family of knob associated tandem repeats, the TR1, which are capable of forming fold-back DNA segments, as well as a new family of centromeric tandem repeats, CentC. Analysis of knob and centromeric DNA segments revealed a complex organization in which blocks of tandemly arranged repeating units are interrupted by insertions of other repeated DNA sequences, mostly represented by individual full size copies of retrotransposable elements. There is an obvious preference for the integration/association of certain retrotransposable elements into knobs or centromere regions as well as for integration of retrotransposable elements into certain sites (hot spots) of the 180-bp repeat. DNA hybridization to a blot panel of eight individual maize chromosome addition lines revealed that CentC, TR1, and 180-bp tandem repeats are found in each of these maize chromosomes, but the copy number of each can vary significantly from about 100 to 25,000. In situ hybridization revealed variation among the maize chromosomes in the size of centromeric tandem repeats as well as in the size and composition of knob regions. It was found that knobs may be composed of either 180-bp or TR1, or both repeats, and in addition to large knobs these repeated elements may form micro clusters which are detectable only with the help of in situ hybridization. The association of the fold-back elements with knobs, knob polymorphism and complex structure suggest that maize knob may be consider as megatransposable elements. The discovery of the interspersion of retrotransposable elements among blocks of tandem repeats in maize and some other organisms suggests that this pattern may be basic to heterochromatin organization for eukaryotes.     

A highly repeated FCP centromeric sequence from chaffinch (Fringilla coelebs: Aves) genome is revealed within interchromosomal connectives during mitosis

A highly repeated FCP (Fringilla coelebs PstI element) sequence was localized by FISH in centromeric regions of all chromosomes of the chaffinch. Besides, FISH signal was found also in interchromosomal connectives linking centromeres of non-homologous chromosomes in mitotic cells. The presence of DNA in the connectives was confirmed by immunostaining with anti-dsDNA antibodies as well as in experiments on nick-translation and random primed labeling in situ. Non-denaturing FISH with FCP probe and random primed labeling of non-denatured chromosomes resulted in fluorescence signal on both centromeres and intercentromeric connectives, thus providing evidence for the availability of single-strand DNA tracts in FCP sequence. It is suggested that the highly repeated FCP centromeric sequence may be respondible for interconnection of mitotic chromosomes and may by involved in nuclear architecture maintenance in the chaffinch.     

Satellite DNA from the Y chromosome of the malaria vector Anopheles gambiae

Satellite DNA is an enigmatic component of genomic DNA with unclear function that has been regarded as "junk." Yet, persistence of these tandem highly repetitive sequences in heterochromatic regions of most eukaryotic chromosomes attests to their importance in the genome. We explored the Anopheles gambiae genome for the presence of satellite repeats and identified 12 novel satellite DNA families. Certain families were found in close juxtaposition within the genome. Six satellites, falling into two evolutionarily linked groups, were investigated in detail. Four of them were experimentally confirmed to be linked to the Y chromosome, whereas their relatives occupy centromeric regions of either the X chromosome or the autosomes. A complex evolutionary pattern was revealed among the AgY477-like satellites, suggesting their rapid turnover in the A. gambiae complex and, potentially, recombination between sex chromosomes. The substitution pattern suggested rolling circle replication as an array expansion mechanism in the Y-linked 53-bp satellite families. Despite residing in different portions of the genome, the 53-bp satellites share the same monomer lengths, apparently maintained by molecular drive or structural constraints. Potential functional centromeric DNA structures, consisting of twofold dyad symmetries flanked by a common sequence motif, have been identified in both satellite groups.     

Genomic organization of alpha satellite DNA on human chromosome 7: evidence for two distinct alphoid domains on a single chromosome.

A complete understanding of chromosomal disjunction during mitosis and meiosis in complex genomes such as the human genome awaits detailed characterization of both the molecular structure and genetic behavior of the centromeric regions of chromosomes. Such analyses in turn require knowledge of the organization and nature of DNA sequences associated with centromeres. The most prominent class of centromeric DNA sequences in the human genome is the alpha satellite family of tandemly repeated DNA, which is organized as distinct chromosomal subsets. Each subset is characterized by a particular multimeric higher-order repeat unit consisting of tandemly reiterated, diverged alpha satellite monomers of approximately 171 base pairs. The higher-order repeat units are themselves tandemly reiterated and represent the most recently amplified or fixed alphoid sequences. We present evidence that there are at least two independent domains of alpha satellite DNA on chromosome 7, each characterized by their own distinct higher-order repeat structure. We determined the complete nucleotide sequences of a 6-monomer higher-order repeat unit, which is present in approximately 500 copies per chromosome 7, as well as those of a less-abundant (approximately 10 copies) 16-monomer higher-order repeat unit. Sequence analysis indicated that these repeats are evolutionarily distinct. Genomic hybridization experiments established that each is maintained in relatively homogeneous tandem arrays with no detectable interspersion. We propose mechanisms by which multiple unrelated higher-order repeat domains may be formed and maintained within a single chromosomal subset.     

Localisation of reiterated nucleotide sequences in Drosophila and mouse by in situ hybridisation of complementary RNA

The location of highly reiterated nucleotide sequences on the chromosomes has been studied by the technique of in situ hybridisation between the DNA of either Drosophila melanogaster salivary gland chromosomes or mouse chromosomes and tritium labelled complementary RNA (c-RNA) transcribed in vitro from appropriate templates with the aid of DNA dependent RNA polymerase extracted from Micrococcus lysodeikticus. The location of the hybrid material was identified by autoradiography after RNase treatment. — When Drosophila c-RNA, transcribed from whole DNA, was annealed with homologous salivary chromosomes in the presence of formamide the well defined labelling was confined to the chromocentre. With heat instead of formamide denaturation there was evidence of discontinuous labelling in various chromosome regions as well, apparently associated with banding. Xenopus ribosomal RNA showed no evidence of annealing to Drosophila chromosomes with the comparatively short exposure times used here. — When mouse satellite DNA was used as template the resulting c-RNA showed no hybridisation to Drosophila chromosomes but, when annealed with mouse chromosomes, the centromeric regions were intensely labelled. The interphase nuclei showed several distinct regions of high activity which suggested aggregation of centromeric regions of both homologous and non-homologous chromosomes. The results of annealing either c-RNA or labelled satellite DNA to homologous chromosomes were virtually indistinguishable. Incubation of Drosophila c-RNA with mouse chromosomes provided no evidence of localisation of grains. — It is inferred that both in mouse and Drosophila the centromeric regions of all chromosomes are enriched in highly reiterated sequences. This may be a general phenomenon and it might be tentatively suggested that the highly reiterated sequences play some role in promoting the close physical approximation of homologous and non-homologous chromosomes or chromosome regions to facilitate regulation of function.