A highly conserved pericentromeric domain in human and gorilla chromosomes.

Significant similarity between human and gorilla genomes has been found in all chromosome arms, but not in centromeres, using whole-comparative genomic hybridization (W-CGH). In human chromosomes, centromeric regions, generally containing highly repetitive DNAs, are characterized by the presence of specific human DNA sequences and an absence of homology with gorilla DNA sequences. The only exception is the pericentromeric area of human chromosome 9, which, in addition to a large block of human DNA, also contains a region of homology with gorilla DNA sequences; the localization of these sequences coincides with that of human satellite III. Since highly repetitive DNAs are known for their high mutation frequency, we hypothesized that the chromosome 9 pericentromeric DNA conserved in human chromosomes and deriving from the gorilla genome may thus play some important functional role.

Whole-comparative genomic hybridization in domestic sheep (Ovis aries) breeds.

Whole-comparative genomic hybridization (W-CGH) allows identification of chromosomal polymorphisms related to highly repetitive DNA sequences localized in constitutive heterochromatin. Such polymorphisms are detected establishing competition between genomic DNAs in an in situ hybridization environment without subtraction of highly repetitive DNA sequences, when comparing two species from closely related taxa (same species, sub-species, or breeds) or somewhat related taxa. This experimental approach was applied to investigating differences in highly repetitive sequences of three sheep breeds (Castellana, Ojalada, and Assaf). To this end, W-CGH was carried out using mouflon (sheep ancestor) chromosomes as a common target to co-hybridize equimolar quantities of two genomic DNAs obtained from either Castellana, Ojalada or Assaf sheep breeds. The results showed that the amount of constitutive heterochromatin is greater in all pericentromeric heterochromatin regions of acrocentric chromosomes than in metacentric or sex chromosomes. Additionally, when W-CGH was performed using DNAs from the Iberian breeds Castellana and Ojalada, chromosomal pericentromeric regions revealed quantitatively and qualitatively a presence of DNA families similar to that obtained from any of the above-cited breeds. On the contrary, when the DNA used in W-CGH experiments was obtained from Assaf, as compared to either Castellana or Ojalada, two different pericentromeric DNA families of highly repetitive sequences could be detected. Lastly, sex chromosomes were shown to be homogeneous among all breeds and thus revealed no detectable constitutive heterochromatin. W-CGH results were confirmed using DNA breakage detection-FISH experiments (DBD-FISH) carried out on lymphocytes. As a whole, the results showed that two different repetitive DNA families are present in the pericentromeric heterochromatin of the sheep breeds studied here. Additionally, they suggest a differential presence of these distinct repetitive DNA families in Castellana and Ojalada breeds as compared to the Assaf breed. Finally, the results of W-CGH after using mouflon as the targeted chromosomes also show that the two DNA families are present in the ancestor.

Some remarks on the use of TaqI to detect highly repetitive DNA sequences in human chromosomes.

In the attempt to conclude investigation of the action of restriction endonucleases on eukaryote chromosomes, we carried out a series of experiments digesting in situ human metaphase chromosomes with AluI/TaqI followed by Giemsa staining. We focused on the centromeric regions of chromosomes1, 2 and 16 and noted that those areas appeared as intensely stained blocks after AluI digestion, but were dramatically reduced in size or completely destroyed after subsequent TaqI treatment. These results permitted us to draw some conclusions on the highly repetitive DNA composition of these regions, in terms of alphoid and classical satellite DNAs.

A cytological approach to the role of guanine in determining quinacrine fluorescence response in eukaryotic chromosomes.

Photooxidation is believed to preferentially remove guanine (G) residues from chromosomal DNA. G interspersion, moreover, has been hypothesized as quenching quinacrine (Q) fluorescence in cytological preparations. Hence, we used photooxidation as a tool for inducing possible changes in the Q-banding pattern of Drosophila melanogaster, Drosophila virilis, and Mus musculus metaphase chromosomes. An enhanced Q fluorescence, which was particularly evident in certain chromosomal regions, was found. This finding would support the postulated primary role of G in determining Q bands in eukaryotic chromosomes.

Selective digestion of human metaphase chromosomes by Alu I restriction endonuclease.

Human cytological preparations have been digested with Alu I restriction endonuclease (Endo R Alu I) and subsequently stained with acridine orange. Metaphase chromosomes treated in this way revealed a clear-cut bright/ dull longitudinal differentiation. It has been postulated that these cytological findings could be related to the selective digestion, by Alu I, of all DNA with the exception of some heterochromatic, possibly satellite, fractions.

Effect of HpaII and MspI restriction endonucleases on chronic myelogenous leukemia chromosomes. Detection of CpG dinucleotide demethylation in situ.

The restriction endonucleases HpaII and MspI both cleave the nucleotide sequence CCGG, but the action of HpaII is inhibited if the internal cytosine is methylated. HpaII and MspI were used on fixed chromosomes from bone marrow cells of individuals suffering from chronic myelogenous leukemia and healthy individuals. We found that MspI acts with the same efficiency on all chromosome samples, whereas HpaII extracts more DNA from the chromosomes of leukemic individuals than from the chromosomes of nonleukemic individuals. We postulate that demethylation of cytosine in the CpG dinucleotide of leukemic cell DNA accounts for our findings.

Scene perception: a failure to find a benefit from prior expectancy or familiarity.

In our everyday world, we typically have an expectancy as to the kinds of scenes that we will see from one glance to the next. Also, many of the scenes that we do see are familiar in the sense that they have been experienced before. Do these factors influence the perception of a scene? In three experiments, priming subjects with a verbal descriptor of a scene was not found to improve reliably the perception of that scene as assessed by the speed and accuracy of detecting an incongruity between an object and its setting (Experiments 1 and 2) or a specified target object (Experiment 3). Also, in attempting to perceive these scenes, subjects could not capitalize on the residue from prior exposures of a scene's background, even though those backgrounds had been processed to the point where semantic information had been extracted from them. Although these results are inconsistent with recent speculations on the role of frames in scene perception, recent experiments on the perception of a scene from a single fixation, and film-editing practice with "flash cuts." The implications of these results are that the mechanisms for perceiving and interpreting nondegraded real-world scenes are so quick and efficient that conditions can readily be found in which priming and prior exposures of substantial portions of scenes are not helpful for perceiving and judging certain aspects of those scenes.

DNA strandness changes during in situ hybridization conditions.

DNA strandness changes occurring during in situ hybridization were monitored using monoclonal antibodies. Our results show that: 1) RNase induces formation of single stranded DNA, 2) after denaturation-renaturation, single stranded DNA is found principally in centromeric areas, and 3) double stranded DNA is still observed after each step.

The organization of classical satellite DNAs in human chromosomes: an approach using AluI and TaqI restriction endonucleases.

Human classical satellite DNAs were used as probes to investigate the molecular mechanism(s) of AluI/TaqI attack in situ on specific centromeric areas. The biochemical results obtained show that the majority of such highly repetitive DNAs are not solubilized from chromosomes, in spite of a cleavage pattern identical to that shown in naked genomic DNA digested with the same enzymes. Moreover, when digestion in situ with restriction enzymes precedes in situ hybridization, it is possible to observe an increased signal in the centromeres of some chromosomes as compared to that shown in standard undigested chromosomes and, on the other hand, hybridization labelling in centromeres which are difficult to detect by in situ hybridization using standard undigested chromosomes. Lastly, our results show that centromeric heterochromatin is not a homogeneous class in regard to organizational structure.

The effects of enzyme inactivation and incubation buffer on digestion in situ with restriction endonucleases.

Previous studies have shown that components of the incubation reaction other than the restriction endonucleases in an in situ restriction enzyme digest of chromosomes may induce G-like banding patterns. To determine whether factors other than DNA base composition play a role in determining restriction enzyme induced bands, we investigated the effect of reaction buffers alone or in the presence of heat inactivated enzymes. Our results show that enzymes such as AluI, RsaI and MspI become inactivated during 3-24 hr incubations at 37 C and that reaction buffers alone failed to produce G-like bands when inactive endonucleases were included.

Chromosomal localisation and genetic variation of the SLC11A1 gene in goats (Capra hircus).

The solute carrier family 11 member A1 (SLC11A1) gene is associated with resistance to infectious diseases. Chromosomal localisation, genomic regions corresponding to functional domains and the genetic variability of microsatellites in the 3' untranslated region (3'-UTR) of this gene were investigated in 427 goats (Capra hircus) of six breeds. Using dual colour fluorescence in situ hybridisation, SLC11A1 was localised to goat chromosome 2. Single strand conformation polymorphism was used to screen for polymorphisms in SLC11A1 exons 2, 10 and 15. There was no variation among goat breeds in the sarcoma homology 3 (SH3) binding motif, the protein kinase C phosphorylation site or the two N-linked glycosylation sites. Exon 15 exhibited variability due to the presence of two polymorphic microsatellites. Genotyping of the upstream guanine-thymine repeat (GTn) at 3'-UTR revealed eight alleles (GT11, GT12, GT14-GT19) in goats, whereas GT13 (present in cattle) was absent. Most goats carried the GT16 allele and no allele was found to be exclusive to only one breed. The coefficient of genetic differentiation value (G(ST)) was 0.084. This microsatellite appears to be an informative DNA marker for genetic linkage analysis in goats.

Identification of two new repetitive elements and chromosomal mapping of repetitive DNA sequences in the fish Gymnothorax unicolor (Anguilliformes: Muraenidae).

Muraenidae is a species-rich family, with relationships among genera and species and taxonomy that have not been completely clarified. Few cytogenetic studies have been conducted on this family, and all of them showed the same diploid chromosome number (2n=42) but with conspicuous karyotypic variation among species. The Mediterranean moray eel Gymnothorax unicolor was previously cytogenetically studied using classical techniques that allowed the characterization of its karyotype structure and the constitutive heterochromatin and argyrophilic nucleolar organizer regions (Ag-NORs) distribution pattern. In the present study, we describe two new repetitive elements (called GuMboI and GuDdeI) obtained from restricted genomic DNA of G. unicolor that were characterized by Southern blot and physically localized by in situ hybridization on metaphase chromosomes. As they are highly repetitive DNA sequences, they map in heterochromatic regions. However, while GuDdeI was localized in the centromeric regions, the GuMboI fraction was distributed on some centromeres and was co-localized with the nucleolus organizer region (NOR). Comparative analysis with other Mediterranean species such as Muraena helena pointed out that these DNA fractions are species-specific and could potentially be used for species discrimination. As a new contribution to the karyotype of this species, we found that the major ribosomal genes are localized on acrocentric chromosome 9 and that the telomeres of each chromosome are composed of a tandem repeat derived from a poly-TTAGGG DNA sequence, as it occurs in most vertebrate species. The results obtained add new information useful in comparative genomics at the chromosomal level and contribute to the cytogenetic knowledge regarding this fish family, which has not been extensively studied.

Shared Y chromosome repetitive DNA sequences in stallion and donkey as visualized using whole-genomic comparative hybridization.

The genome of stallion (Spanish breed) and donkey (Spanish endemic Zamorano-Leonés) were compared using whole comparative genomic in situ hybridization (W-CGH) technique, with special reference to the variability observed in the Y chromosome. Results show that these diverging genomes still share some highly repetitive DNA families localized in pericentromeric regions and, in the particular case of the Y chromosome, a sub-family of highly repeated DNA sequences, greatly expanded in the donkey genome, accounts for a large part of the chromatin in the stallion Y chromosome.

Restriction endonuclease digestion and chromosome banding in the mosquito, Culiseta longiareolata (Diptera: Culicidae).

Fixed chromosomes of the mosquito, Culiseta longiareolata (2n = 6) were treated in situ with nine restriction endonucleases and stained with ethidium bromide or Giemsa. All the heterochromatic regions were apparently protected from digestion by all enzymes except Mbo I. This enzyme selectively digested one of the three types of heterochromatin present in the species. Staining with the fluorochrome quinacrine after enzyme treatment produced a standard Q-banding pattern or a Hoechst 33258-like pattern, depending on the enzyme. These results confirmed: (a) the presence of three types of heterochromatic containing different DNA fractions in the chromosomes of this species, (b) restriction enzymes accessibility to the DNA of heterochromatin regions, and (c) the selective cleavage of particular DNA fractions without DNA removal. Moreover, quinacrine staining after enzyme digestion proved useful in detecting differential activity among enzymes which produced the same banding pattern with standard dyes.

Inter- and intraspecific heterochromatin variation detected by restriction endonuclease digestion in two sibling species of the Anopheles maculipennis complex.

The sibling species Anopheles atroparvus and Anopheles labranchiae are cytogenetically almost indistinguishable. The chromosome complement (2n = 6) consists of two pairs of autosomes and two heteromorphic sex chromosomes with largely homologous heterochromatic long arms. Treatment of chromosome preparations with the restriction endonucleases, Alu I, Hae III, Mbo I, Hpa II, revealed species-specific differences of the sex chromosome banding pattern. These differences involved both amount and location of digested heterochromatin. Heterochromatin heterogeneity and a high level of intraspecific polymorphism, undetected with standard banding techniques, were observed in both species. Quantitative heterochromatin differences between the sex chromosomes did not inhibit their pairing and chiasmata formation. The endonuclease Msp I, which cleaves the same target sequence as Hpa II, did not digest heterochromatic as well as euchromatic regions in both species: inhibition of cleavage by methylation of the target sequence or limited access of the enzyme to the target could be involved in this response.

The effects of aging on fixed DNA.

Agarose-gel electrophoretic analysis was carried out to study the effect(s) of aging on fixed naked DNA as well as on the DNA part of fixed chromosomes. Results show that aging of fixed DNA produces alterations in molecular size; the alteration is more effective in naked DNA than in the DNA part of fixed chromosomes; and the alteration occurs to a greater extent in air-dried DNA than in DNA aged in fixative. Since B-mercaptoethanol is capable of preventing DNA alterations induced by air-drying, oxidative processes are invoked for explaining these findings as well as the effect(s) of aging on fixed cytological preparations.

Characterization and origin of extrachromosomal DNA granules in Sarcophaga bullata.

We have used endonuclease treatment in situ, followed by Giemsa or ethidium bromide staining, for mapping repetitive sequences on the chromosomes of the flesh fly Sarcophaga bullata and thus for studying extrachromosomal DNA granules in this species. All three restriction enzymes employed (HaeIII, A1uI and HindIII) show the same cytological effects, except for a single interstitial band. In both polytene and mitotic chromosomes, chromatin resistant to these endonucleases presumably includes at least three endonucleases presumably includes at least three previously unrecognized buoyant density satellites (1.663, 1.670 and 1.692 g ml-1 in neutral CsCl), and is predominantly localized in the pericentric regions of all five autosomes. Mitotic treated chromosomes show that the entire rod-shaped X chromosome, but no part of the dot-like Y chromosome, consists of endonuclease-resistant chromatin. The most unusual heterochromatic component of polytene nuclei in this species, the 'extrachromosomal DNA granules', are also entirely resistant to digestion with endonucleases. We think that these DNA granules represent dispersed X chromatin and not, as previously assumed, extruded autosomal heterochromatin.

Factors affecting the digestion of restriction endonucleases in situ.

The influence of incubation buffers and glycerol on the enzyme activity of naked DNA of lambda phage and mouse, and of mouse chromosomal DNA was investigated. The results obtained varied in part from previously known data, but confirmed the importance of these factors in determining the patterns of in situ restriction enzyme digestion so far attributed exclusively to endonuclease activity.

Identification of chromosome markers in interphase nuclei.

The chromosome complement of the mosquito Cuilseta longiareolata (2n=6) reveals distinguishable centromeric regions and one telomere of the Y chromosome by using light-induced differentiation and autoradiographic techniques in mitotic and premeiotic interphase nuclei. The localization of these cytological markers and their spatial relationships appear to be very similar in the two types of nuclei and suggest an interphase arrangement where centromeric regions are clustered together in a chromocenter like structure, close to the nuclear membrane, with the telomeres lying on the opposite pole of the nucleus.

In situ digestion of Drosophila virilis polytene chromosomes by AluI and HaeIII restriction endonucleases.

Polytene chromosomes of Drosophila virilis were treated with AluI and HaeIII restriction endonucleases. Both enzymes were capable of extensively digesting chromosomal DNA, with the exception of some regions that contain repetitive DNAs. Moreover, a comparison was made between our data and the data already obtained with the same enzymes in D. melanogaster. On this basis, AluI digestion showed that the 5S RNA genes of D. virilis and D. melanogaster have different base composition, while digestion with HaeIII revealed resistance of the histone genes in D. virilis, contrary to what was previously found in D. melanogaster.