A molecular cytogenetic comparison between Rhynchosciara americana and Rhynchosciara hollaenderi (Diptera: Sciaridae).

The polytene chromosomes of Rhynchosciara americana and R. hollaenderi, a pair of sibling species in the americana-like group of Rhynchosciara, were compared using a number of techniques, including in situ hybridization. With classical cytological techniques, the only differences observed were in the morphology of centromeric and telomeric heterochromatin, in the size of a DNA and RNA puff, and in the presence of an inversion polymorphism in R. hollaenderi. However, after in situ hybridization with rDNA and poly-r(A) probes, differences between the two species appeared at a number of sites. Differences in poly-r(A) sites were especially informative in establishing phylogenetic relationships between these two species and a third species currently being examined from this group. Chromosomal evolution between these species appears to have occurred mainly through differential amplification and transposition of repetitive sequence DNA, of which dA:dT tracts are an important component. The R. hollaenderi karyotype is tentatively considered more ancestral than that of R. americana because it has features present in the third Rhynchosciara species. Explanations for the monomorphisms observed in Rhynchosciara species and mechanisms of speciation in the group are considered within the context of the species' complex behavior.     

Telomeric DNA allocation in chromosomes of common shrew (Sorex araneus, eulipotyphla)

Recently, we displayed an Iberian shrew species (Sorex granarius) with telomere structures unusual for mammals. Long telomeres on the short acrocentric arms contain an average of 213 kb of telomere repeats, whereas the other chromosomal ends have only 3.8 kb (Zhdanova et al., 2005; 2007). However, it is not clear whether these telomeres are typical for all shrew species or only for S. granarius. S. granarius and common shrew Sorex araneus are sibling species. In this study, using modified Q-FISH we demonstrated that telomeres in S. araneus from various chromosomal races distinguished by their number of metacentrics contain 6.8–15.2 kb of telomeric tracts. The S. araneus telomere lengths appear to correspond to telomere lengths in the majority of both shrew species and wild mammals, whereas S. granarius has telomeres with unique or rare structures. Using DNA and RNA high-specific modified probes to telomeric repeats (PNA and LNA), we showed that interstitial telomeric sites in S. araneus chromosomes contain mainly telomeric DNA and that their localization coincide with some evolutionary breakpoints. Interstitial telomeric DNA in S. granarius chromosomes was not revealed. Thus, the distribution of telomeric DNA may be significantly different, even in closely related species whose chromosomes are composed of almost identical chromosomal arms.     

Histone synthesis in Rhynchosciara salivary glands. I. Site and timing of synthesis.

The site of histone synthesis was studied in polytene cells of the salivary glands of the Rhynchosciara americana (Diptera). It was found that, as is the case in non-polytene systems, these proteins are synthesized in the cytoplasm in a class of light polysomes which contain 3-4 ribosomes. This class of polyribosomes is most active at about 5 days before pupation when the nuclei are most active in DNA synthesis and the chromosomes of the gland show many open 'DNA puffs'.     

Species-specific localization of DNA from pericentromeric heterochromatin on polytene chromosomes in the salivary gland cells and 3D-nuclear organization nurse cells in Drosophila virilis and Drosophila kanekoi (Diptera: Drosophilidae)

Microdissection of the chromocenter of D. virilis salivary gland polytene chromosomes has been carried out and the region-specific DNA library (DvirIII) has been obtained. FISH was used for DvirIII hybridization with salivary gland polytene chromosomes and ovarian nurse cells of D. virilis and D. kanekoi. Localization of DvirIII in the pericentromeric regions of chromosomes and in the telomeric region of chromosome 5 was observed in both species. Moreover, species specificity in the localization of DNA sequences of DvirIII in some chromosomal regions was detected. In order to study the three-dimensional organization of pericentromeric heterochromatin region of polytene chromosomes of ovarian nurse cells of D. virilis and D. kanekoi, 3S FISH DvirIII was performed with nurse cells of these species. As a result, species specificity in the distribution of DvirIII signals in the nuclear space was revealed. Namely, the signal was detected in the local chromocenter at one pole of the nucleus in D. virilis, while the signal from the telomeric region of chromosome 5 was detected on another pole. At the same time, DvirIII signals in D. kanekoi are localized in two separate areas in the nucleus: the first belongs to the pericentromeric region of chromosome 2 and another to pericentromeric regions of the remaining chromosomes.     

The chromosomes of Rhynchosciara baschanti (Diptera: Sciaridae): molecular cytogenetic comparisons with taxa in the americana-like group.

Polytene chromosome analysis is presented for Rhynchosciara baschanti, a species belonging to the americana-like group of Rhynchosciara. R. baschanti chromosomes show morphological differences in centromeric and telomeric regions compared to two other members within the group, R. americana and R. hollaenderi. In addition, fixed band and autosomal inversion differences were noted. Physical mapping data showed synteny among the taxa under study for DNA puffs and single-copy or histone gene probes, whereas rDNA and poly-(r)A probes showed different diagnostic patterns. The activity of developmentally active genes and the pattern of thymidine incorporation into DNA puff sites of R. baschanti are consistent with those found in the two previously studied species, except for lower levels of expression at some of these sites. These results suggest that differential duplication of specific DNA sequences, in particular repetitive and homopolymeric DNA, has played a role in the chromosomal evolution of these Rhynchosciara species. Inversions and band dimorphisms have also occurred, but the processes leading to their maintenance and fixation appear to have been slow, since these three species are in general chromosomally monomorphic.     

Homology of Balbiani Ring DNA in two closely related Chironomus species

Cytogenetic analysis indicates that Balbiani Ring 2 (BR 2) in the two sibling species Chironomus tentans and Chironomus pallidivittatus arises from identifically banded segments in the salivary gland polytene chromosomes, although chromosomal rearrangements have occurred. In situ hybridization of BR 2 RNA to the polytene chromosomes of each individual species, as well as their F1 hybrids, reveals that the repetitious BR 2 DNA in the two species has, within the limits of the technique, retained identity of nucleotide sequences and degree of repetition. The DNA of the naturally expressed BR 1 and BR 3 in both species and that ot the galactose induced BR 6 in C. pallidivittatus did not hybridize with BR 2 RNA, indicating that these BR's are different from BR 2 with regard to sequence content.     

Telomeres, interstitial telomeric repeat sequences, and chromosomal aberrations

Telomeres are specialized nucleoproteic complexes localized at the physical ends of linear eukaryotic chromosomes that maintain their stability and integrity. The DNA component of telomeres is characterized by being a G-rich double stranded DNA composed by short fragments tandemly repeated with different sequences depending on the species considered. At the chromosome level, telomeres or, more properly, telomeric repeats--the DNA component of telomeres--can be detected either by using the fluorescence in situ hybridization (FISH) technique with a DNA or a peptide nucleic acid (PNA) (pan)telomeric probe, i.e., which identifies simultaneously all of the telomeres in a metaphase cell, or by the primed in situ labeling (PRINS) reaction using an oligonucleotide primer complementary to the telomeric DNA repeated sequence. Using these techniques, incomplete chromosome elements, acentric fragments, amplification and translocation of telomeric repeat sequences, telomeric associations and telomeric fusions can be identified. In addition, chromosome orientation (CO)-FISH allows to discriminate between the different types of telomeric fusions, namely telomere-telomere and telomere-DNA double strand break fusions and to detect recombination events at the telomere, i.e., telomeric sister-chromatid exchanges (T-SCE). In this review, we summarize our current knowledge of chromosomal aberrations involving telomeres and interstitial telomeric repeat sequences and their induction by physical and chemical mutagens. Since all of the studies on the induction of these types of aberrations were conducted in mammalian cells, the review will be focused on the chromosomal aberrations involving the TTAGGG sequence, i.e., the telomeric repeat sequence that "caps" the chromosomes of all vertebrate species.     

Cytogenetic analysis of Malpighian tubule and salivary gland polytene chromosomes of Bactrocera oleae (Dacus oleae) (Diptera: Tephritidae).

Photomaps of the Malpighian tubule and the salivary gland polytene chromosomes of Bactrocera oleae (Dacus oleae) are presented and compared with those of the fat body. Five polytene chromosomes (10 polytene arms) corresponding to the five autosomes of the mitotic nuclei, as well as a heterochromatic mass corresponding to the sex chromosomes, are observed in the nuclei of the three somatic tissues. The most prominent features of each polytene chromosome, the reverse tandem duplications, as well as the rather unusual ectopic pairing of the telomeric regions of different chromosome arms, are described. The constancy of the banding pattern based on the analysis of the three larval tissues is discussed.     

Mapping the distribution of the interstitial telomeric (TTAGGG)n sequences in eight species of Brazilian marsupials (Didelphidae) by FISH and the correlation with constitutive heterochromatin. Do ITS represent evidence for fusion events in American marsupials?

The C-band pattern and the distribution of the (TTAGGG)(n) sequence after fluorescence in situ hybridization (FISH), were studied in eight species of Didelphidae marsupials: four species with 2n = 14 (Marmosops parvidens, Marmosops incanus, Marmosa murina and Metachirus nudicaudatus), two species with 2n = 18 (Monodelphis domestica and M. americana), and two with 2n = 22 (Didelphis marsupialis and Lutreolina crassicaudata). The hybridization signals were observed at both termini telomeres of all chromosomes. In addition, interstitial sequences were detected in the pericentromeric region of all chromosomes of Marmosops parvidens, in five chromosome pairs of M. incanus, and in the first pair of Monodelphis domestica. These sites always occur in the region of constitutive heterochromatin, even though C-band positive regions do not always present interstitial telomeric sequences (ITS). We suggest that the interstitial (TTAGGG)(n) sequences are associated with satellite DNA and do not necessarily arise through chromosomal rearrangements.     

Intrachromosomal location of the telomeric repeat (TTAGGG)n

Eukaryotic telomeres are specialized DNA-protein structures that are thought to ensure chromosomal stability and complete replication of the chromosome ends. All telomeres which have been studied consist of a tandem array of G-rich repeats which seem to be sufficient for telomere function. Originally, the human telomeric repeat (TTAGGG)_n was assumed to be exclusively located at the very end of all human chromosomes. More recent evidence, however, suggests an extension into proterminal regions. Very little is known about the interstitial distribution of telomeric repeats. Here we present evidence for the presence of (TTAGGG)_n repeats in internal loci on the long and short arms of different human chromosomes. In addition, we studied the genomic organization of these repeats in more detail and discuss possible functions of interstitial telomeric repeats in the human genome.     

Molecular and cytogenetic analysis of the telomeric (TTAGGG)n repetitive sequences in the Nile tilapia,Oreochromis niloticus (Teleostei: Cichlidae)

The majority of chromosomes in Oreochromis niloticus, as with most fish karyotyped to date, cannot be individually identified owing to their small size. As a first step in establishing a physical map for this important aquaculture species of tilapia we have analyzed the location of the vertebrate telomeric repeat sequence, (TTAGGG)n, in O. niloticus. Southern blot hybridization analysis and a Bal31 sensitivity assay confirm that the vertebrate telomeric repeat is indeed present at O. niloticus chromosomal ends with repeat tracts extending for 4-10 kb on chromosomal ends in erythrocytes. Fluorescent in situ hybridization revealed that (TTAGGG)n is found not only at telomeres, but also at two interstitial loci on chromosome 1. These data support the hypothesis that chromosome 1, which is significantly larger than all the other chromosomes in the karyotype, was produced by the fusion of three chromosomes and explain the overall reduction of chromosomal number from the ancestral teleost karyotype of 2n=48 to 2n=44 observed in tilapia.     

Segments of chromosomal DNA from Rhynchosciara americana that undergo additional rounds of DNA replication in the salivary gland DNA puffs have only weak ARS activity in yeast

We have constructed a library of recombinant phage containing DNA from salivary gland chromosomes of Rhynchosciara americana. We have isolated phage from this library that carry sequences homologous to cDNA clones that hybridize in situ to the DNA puffs at the polytene chromosome regions C3 and C8. This has enabled us to demonstrate a 16-fold amplification of the genomic DNA sequences at these regions during DNA-puffing. At the C8 site there is a sequence element that has characteristics of 'scrambled' moderately repetitive DNA. This is located within 3 kb from the gene encoding a 1.95-kb mRNA. We have assayed restriction fragments from the two DNA puffs for Ars activity in yeast. The only strong Ars activity is associated with a part of the moderately repetitive DNA element from the C8 puff which is not present at this site in all animals.     

A repetitive DNA element,associated with telomeric sequences in Drosophila melanogaster,contains open reading frames

He-T sequences are a complex repetitive family of DNA sequences in Drosophila that are associated with telomeric regions, pericentromeric heterochromatin, and the Y chromosome. A component of the He-T family containing open reading frames (ORFs) is described. These ORF-containing elements within the He-T family are designated T-elements, since hybridization in situ with the polytene salivary gland chromosomes results in detectable signal exclusively at the chromosome tips. One T-element that has been sequenced includes ORFs of 1,428 and 1,614 bp. The ORFs are overlapping but one nucleotide out of frame with respect to each other. The longer ORF contains cysteine-histidine motifs strongly resembling nucleic acid binding domains of gag-like proteins, and the overall organization of the T-element ORFs is reminiscent of LINE elements. The T-elements are transcribed and appear to be conserved in Drosophila species related to D. melanogaster. The results suggest that T-elements may play a role in the structure and/or function of telomeres.by W. Hennig     

Telomeric repeat organization--a comparative in situ study between man and rodent

Human, hamster, and mouse chromosomes show both similarities and differences in telomeric organization, detectable with a novel version of the PRINS technique. The differences allowed us to investigate the fate of the telomeres on a chromosome from one species when this chromosome was introduced into the cells of another species. For this purpose, we tested telomeres in cell lines of somatic cell hybrids containing human chromosomes on a rodent background, finding that the telomeres on human chromosomes could not be discriminated from the telomeres on rodent chromosomes. All telomeres in the cell lines were much shorter than the telomeres in normal cells. In the mouse-derived cell lines, half of the mouse chromosomes were fused to other mouse chromosomes at the ends of their short arms. At the points of fusion we were generally unable to detect telomeric signals. In these cell lines, we also found a fraction of chromosomes ends with only one telomeric signal. In chromosomes where both ends showed only one signal, the relative orientation of the signals appeared to be nonrandom with respect to sister chromatids.