Characterization of AluI repeats of zebrafish (Brachydanio rerio).

Two families of repetitive DNA sequences were isolated from the zebrafish genome and characterized. Eight different sequences were sequenced and classified by two standards, their (G + C) composition and their lengths. For convenience, the sequences were first divided into two types. Type I was (A + T)-rich, was repeated approximately 500,000 times, and constituted approximately 5% of the zebrafish genome. Type II was (G + C)-rich, was reiterated approximately 90,000 times, and comprised approximately 0.5% of the genome. Agarose gel electrophoresis of zebrafish DNA cleaved with AluI revealed three distinguishable bands of repetitive fragments: large (approximately 180 bp, designated RFAL), medium (approximately 140 bp, RFAM), and small (approximately 90 bp, RFAS). The RFAL fragments contained both type I and type II sequences. Limited digestion of genomic DNA indicated that RFAL and RFAM were tandemly arranged in the genome, whereas RFAS showed a mixed pattern of both tandem and interspersed repeated arrangements. Although inclusion of a repetitive sequence in a transgenic construct did not appreciably accelerate homologous integration of transgenes into the zebrafish genome, the AluI sequences could facilitate transgene mapping following chromosomal integration.     

Restriction endonuclease banding of rainbow trout chromosomes

Rainbow trout chromosomes were treated with nine restriction endonucleases, stained with Giemsa, and examined for banding patterns. The enzymes AluI, MboI, HaeIII, HinfI (recognizing four base sequences), and PvuII (recognizing a six base sequence) revealed banding patterns similar to the C-bands produced by treatment with barium hydroxide. The PvuII recognition sequence contains an internal sequence of 4 bp identical to the recognition sequence of AluI. Both enzymes produced centromeric and telomeric banding patterns but the interstitial regions stained less intensely after AluI treatment. After digestion with AluI, silver grains were distributed on chromosomes labeled with [³H]thymidine in a pattern like that seen after AluI-digested chromosomes are stained with Giemsa. Similarly, acridine orange (a dye specific for DNA) stained chromosomes digested with AluI or PvuII in patterns resembling those produced with Giemsa stain. These results support the theory that restriction endonucleases produce bands by cutting the DNA at specific base pairs and the subsequent removal of the fragments results in diminished staining by Giemsa. This technique is simple, reproducible, and in rainbow trout produces a more distinct pattern than that obtained with conventional C-banding methods.     

The effect of restriction enzyme digestion of human metaphase chromosomes on C-band variants of chromosomes 1 and 9

Human metaphase chromosomes, fixed on slides, have beent treated with 8 different restriction endonucleases and 29 combinations of 2 restriction enzymes prior to staining with Giemsa. The endonucleases AluI and DdeI and the combinations AluI + DdeI, AluI + HaeIII, AluI + HinfI, and AluI + MboI have then been used to digest metaphase chromosomes of nine individuals with C-band variants of chromosomes 1 or 9, obtained by the CBG technique. The restriction enzyme resistant chromatin of the paracentromeric regions of chromosomes 1 and 9 has been measured and compared with the corresponding CBG-bands. The size of the enzyme resistant chromatin regions depend upon the type of enzyme(s) used. Treatment with AluI + MboI was the only digestion that acted differently on different chromosome pairs. However, within one pair of homologous chromosomes, all digestions revealed the same variations as conventional C-banding.     

Expression of heterochromatin by restriction endonuclease treatment and distamycin A/DAPI staining of Indian muntjac (Muntiacus muntjak) chromosomes

The constitutive heterochromatin of the Indian muntjac (Muntiacus muntjak) was examined following digestion with various restriction endonucleases (AluI, HaeIII, HinfI, and MboI), as well as by selective fluorescence staining with distamycin A plus 4'-6-diamidino-2-phenylindole. Distinct areas within the C-bands were found to have characteristic staining patterns which were more conspicuous in the sex chromosomes. Two dot-like structures resistant to AluI were found in the X and Y1 chromosomes in the same position as the nucleolus organizer regions.     

Karyotypic characterization of Iheringichthys labrosus (Pisces, Pimelodidae): C-, G- and restriction endonuclease banding

Various chromosomal banding techniques were utilized on the catfish, Iheringichthys labrosus, taken from the Capivara Reservoir. C-banding regions were evidenced in telomeric regions of most of the chromosomes. The B microchromosome appeared totally heterochromatic. The restriction endonuclease AluI produced a banding pattern similar to C-banding in some chromosomes; the B microchromosome, when present, was not digested by this enzyme and remained stained. G-banding was conspicuous in almost all the chromosomes, with the centromeres showing negative G-banding. When the restriction endonuclease BamHI was used, most of the telomeres remained intact, while some centromeres were weakly digested. The B chromosome was also not digested by this enzyme. The first pair of chromosomes showed a pattern of longitudinal bands, both with G-banding and BamHI; this was more evident with G-banding. This banding pattern can be considered a chromosomal marker for this population of I. labrosus.     

Prediction of mammalian meiotic synaptic and recombinational behavior of inversion heterozygotes based on mitotic breakpoint data and the possible evolutionary consequences

The karyotype of moose (2n=68) is characterized by very large C-bands close to the centromeres of most chromosomes. The C-banded material represents 40% of the genome. For further characterization of the heterochromatin chromosome spreads were treated with restriction endonucleases and the restriction enzyme (Re) banding pattern was analysed. HaeIII, AluI, MboI, RsaI and HinfI produced informative Re-bands. DdeI induced an even digestion with no banding. Staining with chromomycin A3 produced bright fluorescence in regions corresponding to C-bands. Labeling with BrdUrd during late S phase differentiates four regions in the C banded area. The sequence of these regions from centromere to telomere are: late, early, late and early replicating.The authors propose the existence of five satellite DNA families with distinctive characteristics of G-C and A-T richness and different replication timing, and point out the different clusters for the endonucleases detailed above and their varying location in the chromosomes examined.     

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.     

Electron microscopy and biochemical analysis of mouse metaphase chromosomes after digestion with restriction endonucleases

Electron microscopy (EM) of whole mounted mouse chromosomes, light microscopy (LM), and agarose gel electrophoresis of DNA were used to investigate the cytological effect on chromosomes of digestion with the restriction endonucleases (REs) AluI, HinfI, HaeIII and HpaII. Treatment with AluI produces C-banding as seen by LM, cuts DNA into small fragments, and reduces the density of centromeres and disperses the chromatin of the arms as determined by EM. Treatment with HinfI produces C-banding, cuts DNA into slightly larger fragments than does AluI and increases the density of centromeres and disperses the fibres in the chromosomal arms. Exposure to HaeIII produces G- + C-banding, cuts the DNA into large fragments, and results in greater density of centromeres and reduced density of arms. Finally HpaII digestion produces G-like bands, cuts the DNA into the largest fragments found and results in greater density of centromeres and the best preservation of chromosomal arms detected by EM. These results provide evidence for: (1) REs producing identical effects in the LM (AluI and HinfI) produce different effects in the EM. (2) All enzymes appear to affect C-bands but while REs such as AluI reduce the density of these regions, other enzymes such as HpaII, HaeIII or HinfI increase their density. Conformational changes in the chromatin could explain this phenomenon. (3) The appearance of chromosomes in the EM is related to the action of REs on isolated DNA. The more the DNA is cut by the enzyme, the greater the alteration of the chromosomal ultrastructure.     

The DNA fragments produced by AluI and BstNI digestion of fixed mouse chromosomes

AluI and BstNI restriction endonucleases were used to study cytological and biochemical effects on centromere DNA in fixed mouse chromosomes. These enzymes were employed, as it is known that AluI is incapable of attacking major satellite DNA, contrary to BstNI that is known to cut this DNA fraction into monomers of 234 bp. After digestion in situ, electrophoretic analysis was carried out to characterize the DNA purified (1) from the material remaining on the chromosomes and (2) from the material solubilized from chromosomes. The DNA was then transferred to a nylon filter and ³²P-labelled major satellite DNA was used as a probe for hybridization experiments. Other preparations were simply stained with Giemsa after digestion in situ with AluI and BstNI. Our results show that although restriction endonuclease cleavage primarily depends on DNA base sequence, this factor is not always sufficient to explain nuclease-induced cytological effects. In fact, the structural organization of peculiar regions such as the centromeres of mouse chromosomes might affect cleavage efficiency when restriction enzyme digestion is performed in situ.M.L. Pardue     

Asymmetric staining of Ctenomys chromosomes after treatment with AluI restriction nuclease

After treatment with the endonuclease AluI for 6 or 24 h, chromosomes of two populations of the South American rodent Ctenomys presented an asymmetric banding pattern after Giemsa staining. These asymmetric patterns were chromosome specific (each chromosome of a pair showed different banding pattern) but constant from cell to cell and between homologous chromosomes of the populations analysed. The nature of this peculiar staining is discussed in the light of the interaction between endonucleases and DNA in chromatin of fixed chromosomes.     

Cytological and biochemical characterization of the in situ endonuclease digestion of fixed Tenebrio molitor chromosomes

Cytological and biochemical experiments were undertaken in order to characterize the action of several restriction enzymes on fixed chromosomes of Tenebrio molitor (Coleoptera). EcoRI cuts the satellite DNA of this organism into suunit monomers of 142 bp in naked DNA and acts on fixed chromosomes cleaving and extracting these tandemly repeated sequences present in median centromeric heterochromatin. AluI, in contrast, is unable to attack the satellite sequences but does cut the main band DNA both in naked DNA and in fixed chromosomes. These enzymes therefore permit the in situ localization of satellite DNA or main band DNA in T. molitor. Other enzymes such HinfI or Sau3A do not produce longitudinal differentiation in chromosomes because of the extraction of DNA from satellite and main band DNA regions. In situ hybridization with a satellite DNA probe from T. molitor confirms that the DNA extracted from the chromosomes is the abundant and homogenous highly repeated DNA present in pericentromeric regions. These results plus the analysis of the DNA fractions retained on the slide and solubilized by the action of the restriction enzymes in situ provide evidence that: (a) as an exception to the rule EcoRI (6 bp cutter) is able to produce chromosome banding; (b) the size of the fragments produced by in situ digestion of satellite DNA with EcoRI is not a limiting factor in the extraction; (c) there is a remarkable accord between the action of EcoRI and AluI on naked DNA and on DNA in fixed chromosomes, and (d) the organization of specific chromosome regions seems to be very important in producing longitudinal differentiation on chromosomes.by E.R. Schmidt     

Chromosome banding in the genusPinus

Somatic chromosomes (2n=24) ofPinus luchuensis Mayr at metaphase were observed by fluorescent banding methods with chromomycin A₃ (CMA) and DAPI. CMA-bands appeared at the interstitial and/or proximal regions of nearly all chromosomes. DAPI-bands appeared at the interstitial and/or centromeric regions of nearly all chromosomes, and pairs of DAPI-dots appeared at the centromeric regions. Each homologous pair of chromosomes in the chromosome complement was identified by the CMA and DAPI fluorescent banding patterns. The interstitial CMA-bands were mostly localized at the secondary constrictions of the Feulgen-stained chromosomes. The fluorescent banding pattern ofP. luchuensis was very similar to that ofP. thunbergii, but was different from that ofP. densiflora.     

Individual interphase chromosome domains revealed by in situ hybridization

Summary The position and arrangement of individual chromosomes in interphase nuclei were examined in mouse-human cell hybrids by in situ hybridization of biotinylated human DNA probes. Intense and even labeling of human chromosomes with little background was observed when polyethylene glycol and Tween-20 were included in hybridization solutions. Human interphase chromosomes were separated from each other in the nucleus, and were confined to well localized domains. Hybrid cells with a single human chromosome showed a reproducible position of this chromosome in the nucleus. Some chromosomes appeared to have a characteristic folding pattern in interphase. Optical section as well as electron microscopy of labeled regions revealed the presence of 0.2 m wide fibers in each interphase domain, as well as adjacent, locally extended 500 nm fibers. Such fibers are consistent with previously proposed structural models of interphase chromosomes.     

Characterization of constitutive heterochromatin in Cebus apella (Cebidae, Primates) and Pan troglodytes (Hominidae, Primates): comparison to human chromosomes.

Using G bands, some homologies between the chromosomes of Cebus apella (CAP) and human chromosomes are difficult to establish. To solve this problem, we analyzed these homologies by fluorescence in situ hybridization using human whole chromosome probes (ZOO-FISH). The results indicated that 1) the human probe for chromosome 2 partially hybridizes with CAP chromosomes 13 and 5, 2) the human probe for chromosome 3 partially hybridizes with CAP chromosomes 18 and 20, 3) the human probe for chromosome 9 partially hybridizes with CAP chromosome 19, and 4) the human probe for chromosome 14 hybridizes with the p-terminal and q-terminal regions of CAP chromosome 6. However, none of the human probes employed hybridized with the heterochromatic regions of CAP chromosomes. For this reason, we characterized the heterochromatic regions of CAP chromosomes and of the chromosomes of Pan troglodytes (PTR), to allow comparison between CAP, PTR, and human chromosomes using in situ digestion of fixed chromosomes with the restriction enzymes AluI, HaeIII, and RsaI and by fluorescent staining with DA/DAPI. The results show that 1) centromeric heterochromatin is heterogeneous in the three species studied and 2) noncentromeric heterochromatin is homogeneous within each of the three species, but is different for each species. Thus, centromeric heterochromatin undergoes a higher degree of variability than noncentromeric heterochromatin.     

The molecular characterization of the genome of Muraena helena L. I. Isolation and hybridization of two MboI-restricted DNA fractions.

To investigate the genome of the anguilliform fish Muraena helena at the molecular level we characterized total DNA by agarose gel electrophoresis after cleavage with AluI, HaeIII, MboI, and DdeI restriction endonucleases. Subsequently, we isolated the DNA from two specific electrophoretic fractions to be used as probes for Southern and in situ hybridization experiments. One such fraction showed an electrophoretic pattern typical of highly repetitive DNA localized in the centromeres of many chromosomes. The other fraction was shown to be located in the nucleolar organizer region, partially coincident with 45S rDNA, and to be composed of highly repetitive sequences.     

Localization of the gene encoding human erythroid-potentiating activity to chromosome region Xp11.1----Xp11.4.

We have localized the human gene for erythroid potentiating activity (EPA) to the X chromosome by analysis of its segregation pattern in mouse-human somatic cell hybrids. The EPA gene has been further localized to human chromosome region Xp11.1----Xp11.4 by in situ hybridization of a molecularly cloned EPA genomic fragment to metaphase chromosomes.     

The 1360 bp long basic repeat unit of calf satellite I DNA contains GC rich nucleus of about 140 bp.

Fine melting profiles of calf satellite I DNA and its fragments obtained after digestion with endoR.EcoRI and endoR.AluI nucleases were investigated. It is shown that the 1360 bp basic repeat unit of calf satellite I DNA contains an about 140 bp long GC rich nucleus. It is localized on the 600 bp restriction fragment obtained after digestion of 1360 bp fragment with endoR.AluI nuclease. The main part of satellite I DNA melts as loops between such GC rich nuclei which strongly influence the melting properties of this satellite. There exist significant differences between the thermal stabilities of fragments containing many nuclei, one nucleus and those in which such nucleus is absent.     

The karyotype of the Iberian imperial eagle (Aquila adalberti) analyzed by classical and DNA replication banding.

We report here for the first time the karyotype of the Iberian imperial eagle (Aquila adalberti). All eagles examined had a diploid number of 82 chromosomes and a greater number of microchromosomes (12 pairs) than has been found in all other species of the Accipitridae family. This karyotypic evidence corroborates the recent separation of A. adalberti from A. heliaca on the basis of molecular data. RB-FPG banding induced a specific banding pattern that allowed us to identify homologous chromosome pairs and revealed features about late and early replicating regions. Several chromosome banding techniques (C-, CMA3-, and restriction endonuclease banding and silver staining) were used to characterize the karyotype more accurately. Two GC-rich, late-replicating heterochromatin regions were found in the W chromosome. These regions are AluI resistant and can be used for sex determination in this species. All microchromosomes were heterochromatic, GC rich, and late replicating. Silver staining revealed active nucleolus organizing regions on a pair of microchromosomes that were entirely heterochromatic and stained intensely after CMA3-banding. Different chromosome rearrangements are discussed in order to establish the phylogenetic relationship between A. adalberti and its most closely related species, A. heliaca.     

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.     

Differentiation of anaerobic polycentric fungi by rDNA PCR-RFLP

The suitability of restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA cluster for discriminating two genera of anaerobic polycentric fungi, Orpinomyces and Anaeromyces, was determined. Three PCR-amplified DNA fragments--nuclear small subunit (SSU; 18S rDNA), the nuclear large subunit (LSU; 28S rDNA) and internal transcribed spacer (ITS)--were restricted with endonucleases AluI, DraI, HinfI and MboI. Although the SSU DNA fragment could be restricted successfully by all four enzymes, no differences were observed between restriction patterns of Orpinomyces and Anaeromyces. The most polymorphic restriction pattern between Orpinomyces and Anaeromyces resulted from cleavage of LSU rDNA fragments cut by AluI and HinfI and ITS fragment cut by DraI and HinfI. Genus-specific RFLP patterns were determined for Orpinomyces and Anaeromyces genera; the results showed that the PCR-RFLP analysis of rDNA offers an easy and rapid tool for differentiation of two polycentric genera of anaerobic fungi, which could be hardly separated on the basis of morphology.