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.     

Somatic association of telocentric chromosomes carrying homologous centromeres in common wheat

Summary Measurements of distances between telocentric chromosomes, either homologous or representing the opposite arms of a metacentric chromosome (complementary telocentrics), were made at metaphase in root tip cells of common wheat carrying two homologous pairs of complementary telocentrics of chromosome 1 B or 6 B (double ditelosomic 1 B or 6 B). The aim was to elucidate the relative locations of the telocentric chromosomes within the cell. The data obtained strongly suggest that all four telocentrics of chromosome 1 B or 6 B are spacially and simultaneously co-associated. In plants carrying two complementary (6 B ^S and 6 B ^L) and a non-related (5 B ^L) telocentric, only the complementary chromosomes were found to be somatically associated. It is thought, therefore, that the somatic association of chromosomes may involve more than two chromosomes in the same association and, since complementary telocentrics are as much associated as homologous, that the homology between centromeres (probably the only homologous region that exists between complementary telocentrics) is a very important condition for somatic association of chromosomes. The spacial arrangement of chromosomes was studied at anaphase and prophase and the polar orientation of chromosomes at prophase was found to resemble anaphase orientation. This was taken as good evidence for the maintenance of the chromosome arrangement — the Rabl orientation — and of the peripheral location of the centromere and its association with the nuclear membrane. Within this general arrangement homologous telocentric chromosomes were frequently seen to have their centromeres associated or directed towards each other. The role of the centromere in somatic association as a spindle fibre attachment and chromosome binder is discussed. It is suggested that for non-homologous chromosomes to become associated in root tips, the only requirement needed should be the homology of centromeres such as exists between complementary telocentrics, or, as a possible alternative, common repeated sequences of DNA molecules around the centromere region.Dedicated to Professor Dr. Marcus M. Rhoades on his 70th birthday.     

The organization of DNA in the mitotic and polytene chromosomes of Sciara coprophila

The organization of DNA in the mitotic metaphase and polytene chromosomes of the fungus gnat, Sciara coprophila, has been studied using base-specific DNA ligands, including anti-nucleoside antibodies. The DNA of metaphase and polytene chromosomes reacts with AT-specific probes (quinacrine, DAPI, Hoechst 33258 and anti-adenosine) and to a somewhat lesser extent with GC-specific probes (mithramycin, chromomycin A₃ and anticytidine). In virtually every band of the polytene chromosomes chromomycin A₃ fluorescence is almost totally quenched by counterstaining with the AT-specific ligand methyl green. This indicates that GC base pairs in most bands are closely interspersed with AT base pairs. The only exceptions are band IV-8A3 and the nucleolus organizer on the X. In contrast, quinacrine and DAPI fluorescence in every band is only slightly quenched by counterstaining with the GC-specific ligand actinomycin D. Thus, each band contains a moderate proportion of AT-rich DNA sequences with few interspersed GC base pairs. — The C-bands in mitotic and polytene chromosomes can be visualized by Giemsa staining after differential extraction of DNA and those in polytene chromosomes by the use of base-specific fluorochromes or antibodies without prior extraction of DNA. C-bands are located in the centromeric region of every chromosome, and the telomeric region of some. The C-bands in the polytene chromosomes contain AT-rich DNA sequences without closely interspered GC base pairs and lack relatively GC-rich sequences. However, one C-band in the centromeric region of chromosome IV contains relatively GC-rich sequences with closely interspersed AT base pairs. — C-bands make up less than 1% of polytene chromosomes compared to nearly 20% of mitotic metaphase chromosomes. The C-bands in polytene chromosomes are detectable with AT-specific or GC-specific probes while those in metaphase chromosomes are not. Thus, during polytenization there is selective replication of highly AT-rich and relatively GC-rich sequences and underreplication of the remainder of the DNA sequences in the constitutive heterochromatin.     

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.     

Genetic studies on heterochromatin in Drosophila melanogaster and their implications for the functions of satellite DNA

In Drosophila melanogaster the centromeric heterochromatin of all chromosomes consists almost entirely of several different satellite DNA sequences. In view of this we have examined by genetic means the meiotic consequences of X chromosomes with partial deletions of their heterochromatin, and have found that the amount and position of recombination on each heterochromatically deleted X is substantially different from that of a normal X. It appears that the amount of heterochromatin is important in modifying the centromere effect on recombination. — In all the deleted Xs tested, chromosome segregation is not appreciably altered from that of a nondeleted control chromosome. Thus satellite DNA does not appear to be an important factor in determining the regular segregation of sex chromosomes in Drosophila. Additionally, since X chromosomes with massive satellite DNA deficiencies are able to participate in a chromocenter within salivary gland nuclei, a major role of satellite DNA in chromocenter formation in this tissue is also quite unlikely. — In order to examine the mechanisms by which the amount of satellite DNA is increased or decreased in vivo, we have measured cytologically the frequency of spontaneous sister chromatid exchanges in a ring Y chromosome which is entirely heterochromatic and consists almost exclusively of satellite DNA. In larval neuroblast cells the frequency of spontaneous SCE in this Y is approximately 0.3% per cell division. Since there is no meiotic recombination in D. melanogaster males and since meiotic recombination in the female does not occur in heterochromatin, our results provide a minimum estimate of the in vivo frequency of SCE in C-banded heterochromatin (which is predominantly simple sequence DNA), without the usual complications of substituted base analogs, incorporated radioactive label or substantial genetic content. — We emphasise that: (a) satellite DNA is not implicated in any major way in recognition processes such as meiotic homologue recognition or chromocenter formation in salivaries, (b) there is likely to be continuous variation in the amount of satellite DNA between individuals of a species; and (c) the amount of satellite DNA can have a crucial functional role in the meiotic recombination system.     

Isolation and partial characterization of replication intermediates in Chironomus polytene chromosomes

DNA replication was investigated in cells with polytene chromosomes. The cells were obtained from the salivary glands of the dipteran Chironomus tentans. Polytene chromosomes are characterized by a specific and constant band — interband structure formed by the lateral association of homologous chromatids side by side. — The salivary gland DNA was labelled by injection of radioactive precursor into the living animal, extracted with a neutral nondenaturing buffer at 25° C and finally characterized by agarose gel electrophoresis. Radioactive DNA pulse-labelled for 30–60 min was released from the polytene chromosomes during cell lysis in the form of double-stranded fragments. The fragments, which show a heterogeneous appearance in gel electrophoresis, are probably produced in the living cell by the joining of several Okazaki fragments. The release of the fragments from the polytene chromosome is prevented by lysis at 0° C instead of 25° C. The size of the double-stranded fragments range between 3.75–6×10⁶ D. Moreover, after a time-lag the fragments are joined together to produce a high-molecular weight DNA. The existence of these nascent DNA fragments is discussed in relation to an earlier proposal that each band in the polytene chromosome may function as a separate replication unit.     

Differential staining of polytene chromosome bands in Chironomus by Giemsa banding methods

Two Giemsa banding methods (C banding and RB banding) are described which selectively stain the centromere bands of polytene salivary gland chromosomes in a number of Chironomus species. — By the C banding method the polytene chromosome appearance is changed grossly. Chromosome bands, as far as they are identifiable, are stained pale with the exception of the centromere bands and in some cases telomeres, which then are intensely stained reddish blue. — By the RB method the centromere bands are stained bright blue, whereas the remainder of the polytene bands stain red to red-violet. — Contrary to all other species examined, in Chironomus th. thummi numerous interstitial polytene chromosome bands, in addition to the centromere regions, are positively C banded and blue stained by RB banding. In the hybrid of Ch. th. thummi x Ch. th. piger only those interstitial thummi bands which are known to have a greater DNA content than their homologous piger bands are C banding positive and blue stained by the RB method whereas the homologous piger bands are C banding negative and red stained by RB banding. Ch. thummi and piger bands with an equal amount of DNA both show no C banding and stain red by RB banding. — It seems that the Giemsa banding methods used are capable of demonstrating, in addition to centromeric heterochromatin, heterochromatin in those interstitial polytene chromosome bands whose DNA content has been increased during chromosome evolution.     

The influence of DNA content and nuclear volume on the frequency of radiation-induced chromosome aberrations in Bufo species

The frequency of chromosome aberrations was compared in X-irradiated blood lymphocytes of Bufo marinus, B. calamita and B. pardalis which have similar chromosome numbers (2n=22) and karyotypes but differ in chromosomal DNA content and nuclear volume. — For each species the yield, per cell of centric exchange aberrations (dicentrics+rings) and of deletions (interstitial and terminal) increased approximately as the 1.5th power of the dose. — The 55% higher chromosomal DNA content of B. pardalis compared with both B. marinus and B. calamita resulted in the same increase in the frequency of deletions as a 55% increase in radiation dose, approximately doubling the yield. Both factors probably lead to a similar increase in the frequency of primary lesions from which the deletions are derived. — In contrast, an increase in chromosomal DNA content did not result in a higher yield of dicentric and ring exchanges, probably because the greater nuclear volume of B. pardalis (twice that of the other species) offset the potential increase in exchanges by increasing the average distance between chromosomes and chromosome arms. The data support the hypothesis that, in order to be involved in exchange, chromosome regions must be close together at the time of irradiation. The frequency of deletions is unaffected by changes in nuclear volume.     

Analysis of Y chromosome heterochromatin in Rumex thyrsiflorus

The Y chromosome heterochromatin in Rumex thyrsiflorus has been analyzed. In natural populations the Y chromosome shows a higher morphological variability than the X chromosome. The total duration of replication of Y chromosomes is about 2 hrs longer than that of euchromatin. Autoradiography with tritiated thymidine showed that chromocentres formed by Y chromosomes in interphase nuclei retain their heterochromatic form during DNA replication. — Y chromosome heterochromatin in interphase nuclei is stained pink, while the rest of the nucleus stains green after fast green-eosin staining for histones. — During the premeiotic stage of PMC development Y chromosomes are no longer visible as compact bodies and become more fuzzy in appearance. A diffuse state of Y coincides with intense RNA synthesis. Therefore genetic activity of Y chromosomes or their parts during premeiotic stage of microsporogenesis is postulated.     

A specific chromosome element,the telomere of Drosophila polytene chromosomes

The submicroscopic organization of terminal chromosome regions of Drosophila hydei polytene chromosomes is described. A compact region composed of tightly packed fibrils of 100 to 125 Å diameter embedded in an amorphous material is located at each of the chromosome ends of the 5 long chromosome arms. From this compact region, sometimes containing cavities, fibrils extend onto the nearest normal band region. The diameter of the extending fibrils is 100–125 Å, 200–250 Å or 400 Å. Pronase digestion of fixed and squashed chromosomes reduced the electron density of the amorphous matrix in the compact regions but failed to affect the diameter of the fibrils. The extending fibrils, however, showed a decrease in diameter after pronase digestion. The most frequently observed diameter values were 100–125 Å. — The volume of the terminal structures, including the compact region as well as the extending fibrils, is characteristically different for the various elements of the karyotype. Chromosome 2 displays the largest terminal structure, whereas chromosome 4 only occasionally shows the presence of compact regions. — End to end association of the long chromosome arms involves the fusion of the compact terminal structures. The non-random distribution of end to end association seems to be correlated with the volume of the terminal structures. Chromosome 2 which contains the largest compact terminal region is more frequently involved in end to end associations than any other chromosome arm. — The terminal regions show replication of DNA. They belong to the group of regions which display a discontinuous labeling pattern along the chromosomes, representing a late phase of the replication cycle. — The unique structural organization of the terminal chromosome regions, which is never observed at any other location of the genome supports the idea that they are morphological manifestations of the postulated telomeres.     

Molecular-cytogenetic characterization of a higher plant centromere/kinetochore complex

The centromeric region of a telocentric field bean chromosome that resulted from centric fission of the metacentric satellite chromosome was microdissected. The DNA of this region was amplified and biotinylated by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR)/linker-adapter PCR. After fluorescence in situ hybridization (FISH) the entire chromosome complement of Vicia faba was labelled by these probes except for the nucleolus organizing region (NOR) and the interstitial heterochromatin, the chromosomes of V. sativa and V. narbonensis were only slightly labelled by the same probes. Dense uniform labelling was also observed when a probe amplified from a clearly delimited microdissected centromeric region of a mutant of Tradescantia paludosa was hybridized to T. paludosa chromosomes. Even after six cycles of subtractive hybridization between DNA fragments amplified from centromeric and acentric regions no sequences specifically located at the field bean centromeres were found among the remaining DNA. A mouse antiserum was produced which detected nuclear proteins of 33 kDa and 68 kDa; these were predominantly located at V. faba kinetochores during mitotic metaphase. DNA amplified from the chromatin fraction adsorbed by this serum out of the sonicated total mitotic chromatin also did not cause specific labelling of primary constrictions. From these results we conclude: (1) either centromere-specific DNA sequences are not very conserved among higher plants and are — at least in species with large genomes — intermingled with complex dispersed repetitive sequences that prevent the purification of the former, or (2) (some of) the dispersed repeats themselves specify the primary constrictions by stereophysical parameters rather than by their base sequence.     

Mitotic inter-homologue junctions accumulate at damaged DNA replication forks in recQ mutants

Mitotic homologous recombination is utilised to repair DNA breaks using either sister chromatids or homologous chromosomes as templates. Because sister chromatids are identical, exchanges between sister chromatids have no consequences for the maintenance of genomic integrity unless they involve repetitive DNA sequences. Conversely, homologous chromosomes might differ in genetic content, and exchanges between homologues might lead to loss of heterozygosity and subsequent inactivation of functional genes. Genomic instability, caused by unscheduled recombination events between homologous chromosomes, is enhanced in the absence of RecQ DNA helicases, as observed in Bloom's cancer-prone syndrome. Here, we used two-dimensional gel electrophoresis to analyse budding yeast diploid cells that were modified to distinguish replication intermediates originating from each homologous chromosome. Therefore, these cells were suitable for analysing the formation of inter-homologue junctions. We found that Rad51-dependent DNA structures resembling inter-homologue junctions accumulate together with sister chromatid junctions at damaged DNA replication forks in recQ mutants, but not in the absence of Srs2 or Mph1 DNA recombination helicases. Inter-homologue joint molecules in recQ mutants are less abundant than sister chromatid junctions, but they accumulate with similar kinetics after origin firing under conditions of DNA damage. We propose that unscheduled accumulation of inter-homologue junctions during DNA replication might account for allelic recombination defects in recQ mutants.     

Location of the genes for 5S ribosomal RNA in Xenopus laevis

In situ hybridization of 5S RNA and cRNA transcribed in vitro from Xenopus laevis 5S DNA shows that 5S DNA is localized at or near the telomere region of the long arm of many, if not all, of the X. laevis chromosomes. No 5S DNA is detected near the nucleolus organizer in the normal X. laevis chromosome complement, but in a X. laevis kidney cell line, 5S DNA is found at the distal end of the secondary constriction. The arrangement of 5S DNA in several types of interphase nuclei is described. — During the pairing stages of meiosis the telomeres of most or perhaps all of the chromosomes become closely associated so that the regions containing 5S DNA form a single cluster. This close association might be either a cause or a result of the presence of the similar sequences of 5S DNA on many telomeres. It suggests that the uniformity of 5S sequences on non-homologous chromosomes might be maintained by crossing-over between the chromosomes.     

Restriction endonuclease and molecular analyses of three rat genomes with special reference to chromosome rearrangement and speciation problems

When differences are found between related species of organisms, it is often assumed that the differences themselves are causal factors either in speciation itself or in processes related to speciation. Two recent proposals on the functions of satellite DNA (Hatch et al., 1976 and Fry and Salser, 1977) are that (a) large amounts of satellite DNA are important in facilitating chromosome rearrangements and hence cytogenetic evolution, and (b) satellite DNA differences between homologous chromosomes lead to pairing difficulties and are important in generating infertility barriers and hence speciation. If these proposals were to have some generality, one could expect organisms with very low amounts of highly repeated DNA to exhibit few chromosome rearrangements and to be evolutionarily conservative in a cyto-genetic sense. — We have chosen two very closely related species of rat which are phenotypically almost indistinguishable and which have undergone massive genome reorganization. They differ by 11 major centric rearrangements (2n=32, 2n=50). We have characterised their genomes by restriction endonuclease digestions, thermal denaturations, analytical ultracentrifugations and reassociation techniques, and have found that they have virtually no highly repeated DNA. Thus the 11 major chromosomal rearrangements have been fixed in present day genomes with hardly any highly repeated DNA, centric or otherwise. — It appears therefore that a large amount of highly repeated DNA is not obligatory for the formation and fixation of chromosome rearrangements. In addition, the existing literature reveals that one can find almost any situation at all, from species groups with high amounts of satellite DNA and no gross chromosomal rearrangements, to ones such as those described here, with tiny amounts of highly repeated DNA and massive chromosomal reorganisation. Since direct experimental data indicates that satellite DNA differences per se between homologous chromosomes do not cause infertility, speculations concerning modes of speciation based on satellite DNA differences between otherwise homologous chromosomes would appear to be ill founded.     

Differences in DNA composition along mammalian metaphase chromosomes

Denaturation of chromosomal DNA in situ can be achieved without disruption of chromosomal morphology by heating slides at 25–90° C in 10–95% formamide in SSC. The extent of denaturation is proportional to formamide concentration and temperature. Reassociation of denatured DNA is prevented with formaldehyde. — The DNA in the paracentromeric constrictions in human chromosomes 1, 9 and 16 denatures earlier than in any other regions, as shown by the red colour with acridine orange. When the temperature or formamide concentration is raised a red and green banding pattern emerges in which regions known to stain brightly with quinacrine mustard are red whereas other regions are green. The last regions to turn red are the short arms of some acrocentric chromosomes. Since A+T-rich DNA denatures before G+C-rich DNA, it is inferred that QM-bright areas are rich in A+T. Similar results are obtained with mouse and Microtus agrestis cells. — Reassociation of chromosomal DNA denatured by heat and formamide occurs if no formaldehyde is used. In human cells, kinetic studies on reassociation indicate that the highest degree of repetition is in the DNA of the distal half of the Y chromosome. Next in degree of repetition are the paracentromeric constrictions, the short arm regions of some of the acrocentric chromosomes, and all the centromeric regions. Highly repetitious DNA is found in all mouse centromeric regions except that of the Y chromosome. Constitutively heterochromatic segments of X and Y and the autosomal centromeric regions of Microtus agrestis also contain repetitious DNA. — It is proposed that differential base content and susceptibility to denaturation of DNA contribute to or at least accompany Q-, G- and R-banding. The degree of C-banding is related to repetitious DNA. The human Y chromosomal DNA is probably A+T-rich and exceptionally repetitious, exhibiting spontaneous reassociation under many experimental conditions.     

SINE and LINE within human centromeres

A number of the Alu and Ll elements present within the centromeric regions of the human chromosomes have been analyzed by polymerase chain reaction amplification. The oligonucleotide primers were homologous to the 3 end consensus sequences of either Alu or Ll in conjunction with an oligonucleotide primer homologous to alphoid sequences specific to different chromosomes. This allowed one to detect an unusual number of Alu and Ll polymorphisms at different loci. It is proposed that this results from molecular rearrangements which occur within the -satellite DNA in which they are embedded (Marçais et al. J. Mol. Evol. 33:42–48, 1991) and not because the centromeric regions are targets for new insertions of such elements. The same analyses were made on cosmids and YACs originating from the centromeric region of chromosome 21 as well as on a collection of somatic hybrids containing chromosome 21 centromere as unique common human genetic material. The results were consistent with the above hypothesis. Correspondence to: G. Roizès     

Sex chromosome associated satellite DNA: Evolution and conservation

Satellites visible in female but not in male DNA were isolated from the snakesElaphe radiata (satellite IV, p = 1.708 g · cm^–3) andBungarus fasciatus (BK¹ minor, p=1.709 g · cm^–3). The satellites cross hybridize. Hybridization of³H labelled nick translated BK minor satellite DNA with the total male and female DNA and/or chromosomes in situ of different species of snakes revealed that its sequences are conserved throughout the snake group and are mainly concentrated on the W chromosome. Snakes lacking sex chromosomes do possess related sequences but there is no sex difference and visible related satellites are absent. The following conclusions have been reached on the basis of these results. 1. The W chromosome associated satellite DNA is related to similar sequences scattered in the genome. 2. The origin and increment in the number of the W satellite DNA sequence on the W chromosome is associated with the heterochromatinization of the W. 3. Satellite sequences have become distributed along the length of the W and resulted in morphological differentiation of sex chromosomes. 4. Evolutionary conservation of W satellite DNA strongly suggests that functional constraints may have limited sequence divergence.     

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.     

Acridine-orange differential fluorescence of fast- and slow-reassociating chromosomal DNA after in situ DNA denaturation and reassociation

A cytological technique based on heat denaturation of in situ chromosomal DNA followed by differential reassociation and staining with acridine orange was developed. Mouse nuclei and chromosomes in fixed cytological preparations show a red-orange fluorescence after thermal DNA denaturation (2–4 minutes at 100° C), and fluoresce green if denaturation is followed by a total DNA reassociation (two minutes or more at 65–66°C). — A reassociation time between a few and 60–90 seconds demonstrates the centromeric heterochromatin of chromosomes (which sometimes aggregate in the form of clusters) and the interphase chromocenters in green, the chromosomal arms fluorescing red-orange. Under the same conditions, the Y chromosome presents a pale green or yellow-green fluorescence along its chromatids, but its centromeric region fluoresces weakly. — The interpretation is suggested that the fast-reassociating chromosomal DNA (as detected by AO in centromeric heterochromatin and interphase chromocenters), represents repetitive DNA.     

Structure and organization of rhodophyte and chromophyte plastid genomes: implications for the ancestry of plastids

Summary Recombinational repair is the means by which DNA double-strand breaks (DSBs) are repaired in yeast. DNA divergence between chromosomes was shown previously to inhibit repair in diploid G1 cells, resulting in chromosome loss at low nonlethal doses of ionizing radiation. Furthermore, 15–20% divergence prevents meiotic recombination between individual pairs of Saccharomyces cerevisiae and S. carlsbergensis chromosomes in an otherwise S. cerevisiae background. Based on analysis of the efficiency of DSB-induced chromosome loss and direct genetic detection of intragenic recombination, we conclude that limited DSB recombinational repair can occur between homoeologous chromosomes. There is no difference in loss between a repair-proficient Pms⁺ strain and a mismatch repair mutant, pms1. Since DSB recombinational repair is tolerant of diverged DNAs, this type of repair could lead to novel genes and altered chromosomes. The sensitivity to DSB-induced loss of 11 individual yeast artificial chromosomes (YACs) containing mouse or human (chromosome 21 or HeLa) DNA was determined. Recombinational repair between a pair of homologous HeLa YACs appears as efficient as that between homologous yeast chromosomes in that there is no loss at low radiation doses. Single YACs exhibited considerable variation in response, although the response for individual YACs was highly reproducible. Based on the results with the yeast homoeologous chromosomes, we propose that the potential exists for intra- YAC recombinational repair between diverged repeat DNA and that the extent of repair is dependent upon the amount of repeat DNA and the degree of divergence. The sensitivity of YACs containing mammalian DNA to ionizing radiation-induced loss may thus be an indicator of the extent of repeat DNA.