DNA methylation in insects.

Cytosine DNA methylation has been demonstrated in numerous eukaryotic organisms and has been shown to play an important role in human disease. The function of DNA methylation has been studied extensively in vertebrates, but establishing its primary role has proved difficult and controversial. Analysing methylation in insects has indicated an apparent functional diversity that seems to argue against a strict functional conservation. To investigate this hypothesis, we here assess the data reported in four different insect species in which DNA methylation has been analysed more thoroughly: the fruit fly Drosophila melanogaster, the cabbage moth Mamestra brassicae, the peach-potato aphid Myzus persicae and the mealybug Planococcus citri.

Facultative heterochromatization in parahaploid male mealybugs: involvement of a heterochromatin-associated protein.

The behavior of chromosomes during development of the mealybug Planococcus citri provides one of the most dramatic examples of facultative heterochromatization. In male embryos, the entire haploid paternal chromosome set becomes heterochromatic at mid-cleavage. Male mealybugs are thus functionally haploid, owing to heterochromatization (parahaploidy). To understand the mechanisms underlying facultative heterochromatization in male mealybugs, we have investigated the possible involvement of an HP-1-like protein in this process. HP-1 is a conserved, nonhistone chromosomal protein with a proposed role in heterochromatinization in other species. It was first identified in Drosophila melanogaster as a protein enriched in the constitutive heterochromatin of polytene chromosome. Using a monoclonal antibody raised against the Drosophila HP-1 in immunoblot and immunocytological experiments, we provide evidence for the presence of an HP-1-like in Planococcus citri males and females. In males, the HP-1-like protein is preferentially associated with the male-specific heterochromatin. In the developing male embryos, its appearance precedes the onset of heterochromatization. In females, the HP-1-like protein displays a scattered but reproducible localization pattern along chromosomes. The results indicate a role for an HP-1-like protein in the facultative heterochromatization process.

The relationship between DNA methylation and chromosome imprinting in the coccid Planococcus citri.

The phenomenon of chromosome, or genomic, imprinting indicates the relevance of parental origin in determining functional differences between alleles, homologous chromosomes, or haploid sets. In mealybug males (Homoptera, Coccoidea), the haploid set of paternal origin undergoes heterochromatization at midcleavage and remains so in most of the tissues. This different behavior of the two haploid sets, which depends on their parental origin, represents one of the most striking examples of chromosome imprinting. In mammals, DNA methylation has been postulated as a possible molecular mechanism to differentially imprint DNA sequences during spermatogenesis or oogenesis. In the present article we addressed the role of DNA methylation in the imprinting of whole haploid sets as it occurs in Coccids. We investigated the DNA methylation patterns at both the molecular and chromosomal level in the mealybug Planococcus citri. We found that in both males and females the paternally derived haploid set is hypomethylated with respect to the maternally derived one. Therefore, in males, it is the paternally derived hypomethylated haploid set that is heterochromatized. Our data suggest that the two haploid sets are imprinted by parent-of-origin-specific DNA methylation with no correlation with the known gene-silencing properties of this base modification.

The base analog 6-N-hydroxylaminopurine (HAP) mutagenesis is dependent on the integrity of the uvsE, uvsF and uvsB genes in Aspergillus nidulans.

Most of the available data in lower eukaryotes are consistent with the idea that base analogs-induced mutagenesis is due to the mis-pairing properties of these compounds, which, in turn, is due to a shift in the tautomeric equilibrium of the molecule. A tautomeric shift may in fact lead to mismatches which, at least in Escherichia coli, can be repaired by genes involved in the post-replicative mismatch repair whose activity is necessary to control spontaneous mutagenesis. In filamentous fungi, such as Aspergillus nidulans, nothing is known about the repair of base pairing mistakes after base analogs treatment. For this reason, we have decided to screen UV-sensitive Aspergillus nidulans mutants for their mutagenic response to 6-N-hydroxylaminopurine (HAP). We have shown that three mutations (uvsB, uvsC and uvsE), which enhance the UV-sensitivity of germinating conidia, cause a lower mutagenic response to HAP. On the other hand, the uvsH mutation, has no effect on HAP-induced mutagenesis.

DNA methylation changes during mouse spermatogenesis.

Genomic imprinting in mammals is thought to be mediated by differences in the methylation level of cytosine residues in the genome. These differences in DNA methylation are thought to be generated during the development of the germ line. To characterize the profile of global methylation of the mouse genome during male gametogenesis, we have quantified the relative level of methylation in individual cells during meiosis and spermatogenesis. A decrease in the level of DNA methylation is observed from meiotic cells to elongated spermatids. The erasure of the somatic pattern of methylation during spermatogenesis suggests the existence of a subsequent mechanism generating the parental specific methylation patterns leading to genomic imprinting of specific alleles.

In human chromosomes telomeric regions are enriched in CpGs relative to R-bands.

Human chromosomes were in situ nick-translated using as nicking agents the endonucleases MspI (CCGG), its methyl-sensitive isoschizomer HpaII, HaeIII (GGCC), SacII (CCGCGG), EcoRI (GAATTC) and DNaseI. We show that in metaphase chromosomes R-bands are enriched, as compared with G-bands, in the dinucleotide CpG but no more than what is expected on the basis of their relative G+C content. The telomeric regions, on the contrary, besides having a chromatin conformation that is particularly relaxed and accessible to endonucleases, also show an enrichment in CpGs.

The human Y chromosome shows a low level of DNA polymorphism.

Six new Y-specific probes have been isolated and are reported. Along with another six already described they have been used in a systemic search for male specific RFLPs. An overall number of 46515 nucleotides have been screened with 12 enzymes and no polymorphic pattern observed. Our data reveal a greatly reduced level of polymorphism compared with other chromosomes.

Analysis of methylation and distribution of CpG sequences on human active and inactive X chromosomes by in situ nick translation.

In situ nick translation of fixed mitotic chromosomes after HpaII or MspI digestion allows us to detect different DNA methylation levels along chromosomes. We used this technique to analyse the methylation levels of CCGG sites in the active and inactive X chromosomes of female human cells. In addition, we analysed the distribution of these sites with respect to the banding pattern. Our data show that the inactive X, as a whole, is more methylated than the active one and that CCGG sequences are preferentially located on R-positive bands.

Human NORs show correlation between transcriptional activity, DNase I sensitivity, and hypomethylation.

Active human ribosomal gene clusters (NORs) are distinguishable from inactive ones by silver staining. By sequentially applying deoxyribonuclease I (DNase I)-directed in situ nick-translation and silver staining to fixed chromosome preparations, we found that active NORs are more sensitive to DNase I than inactive ones. Use of the two restriction isoschizomeres MspI and HpaII to modify the nick-translation technique showed that active NORs are significantly less methylated than inactive ones. Taken as a whole, our results indicate that ribosomal gene activity, DNase I sensitivity, and DNA methylation are closely interrelated.

Screening for cytogenetic polymorphisms in a random sample of liveborn infants from Italian population.

The frequency of major and minor chromosome variants is studied in a random sample of newborns in Central Italy. Special attention is paid to the objective criteria used to evaluate minor variants. In our sample, the frequency of acrocentric chromosome variants is found to be unusually high compared with previous studies. Also, the distribution of C-band sizes differs from that reported for other populations, while the frequency of major chromosome variants is found to be the same.

Simultaneous production of Q and R bands after staining with chromomycin A3 or olivomycin.

Human and mouse chromosomes, stained with either chromomycin A3 or olivomycin, which bind preferentially to G - C-rich DNA (where G is guanosine and C is cytosine), exhibit a Q or a reverse banding pattern, depending on the wavelength used for excitation. The two complementary banding patterns can be observed in the same metaphase simply by changing the combination of excitation filters. These data suggest, therefore, that in addition to base composition, other factors are involved in the production of chromosome banding by chromomycin A3 and olivomycin.

Effects of distamycin A on human leukocytes in vitro.

Distamycin A, an oligopeptide antibiotic, supplied at various concentrations for 24 h to human leukocytes in culture, has induced the appearance on some chromosomes of specific areas lacking spiralization. In particular, the centromeric regions of chromosomes 1, 3 and one C-group chromosome and the distal part of the long arm of the Y chromosome were despiralized. The possible nature of these regions is discussed.

Effects of DAPI on human leukocytes in vitro.

DAPI (4'-6-diamidino-2-phenylindole), a fluorochrome specific for AT-rich DNA, was supplied for 24 h at various concentrations to human leukocytes in culture. This treatment caused the appearance on the chromosomes of specific areas lacking spiralization. In particular, the centromeric regions of chromosomes 1,9, and 16, a short region on the long arm of chromosomes 1 and 2, and the distal heterochromatic part of the long arm of the Y chromosome were despiralized. The despiralization pattern of DAPI is compared with those previously obtained with Hoechst 33258 and Distamycin A.

N-banding and nucleolus organisers in Asellus aquaticus (Crust. Isop.).

The N-banding technique was used to stain the nucleolus organiser of the karyotype of Asellus aquaticus (crust. Isop.). Observations were made on the morphological expression of nucleolus organisers as secondary constrictions and the presence of nucleoli in mitotic prophase. An attempt was made to correlate the various results and it seems likely that N-banding is not a reflection of NO activity.

Effect of Hoechst 33258 on Chinese hamster chromosomes.

Cells of the Chinese hamster strain C-125 were treated for different time intervals with H 33258, a bibenzimidazole derivative. The same compound was used to stain fixed cells of the same strain. H 33258 induced in cells in culture specific areas of reduced spiralization on the metaphase chromosomes of some cells. These probably correspond to DNA segments rich in A-T bases interspersed along the chromosomes. Probably H 33258 acts during S period of cell cycle. The banding obtained by staining with H 33258 is similar to that induced by quinacrine dihydrochloride but shows a better resolution.

Effects of Hoechst 33258 on human leukocytes in vitro.

The benzimidazole derivative Hoechst 33258 was added at various concentrations to human leukocyte cultures. After 16 or 24 h of treatment, with concentrations equal to or greater than 100 mug/ml of Hoechst 33258, a number of chromosomes showed regions in which the chromatin was undercontracted. The centromeric regions of chromosome 1 and, more rarely, of chromosomes 3 and 9 appeared to be decondensed. Short decondensed regions were also present on the long arms of chromosomes 1 and 2. The possible nature of these regions is discussed.