Molecular differentiation of sex chromosomes probed by comparative genomic hybridization

Comparative genomic hybridization (CGH) was used to identify and probe sex chromosomes in several XY and WZ systems. Chromosomes were hybridized simultaneously with FluorX-labelled DNA of females and Cy3-labelled DNA of males in the presence of an excess of Cot-1 DNA or unlabelled DNA of the homogametic sex. CGH visualized the molecular differentiation of the X and Y in the house mouse, Mus musculus, and in Drosophila melanogaster: while autosomes were stained equally by both probes, the X and Y chromosomes were stained preferentially by the female-derived or the male-derived probe, respectively. There was no differential staining of the X and Y chromosomes in the fly Megaselia scalaris, indicating an early stage of sex chromosome differentiation in this species. In the human and the house mouse, labelled DNA of males in the presence of unlabelled DNA of females was sufficient to highlight Y chromosomes in mitosis and interphase. In WZ sex chromosome systems, the silkworm Bombyx mori, the flour moth Ephestia kuehniella, and the wax moth Galleria mellonella, the W chromosomes were identified by CGH in mitosis and meiosis. They were conspicuously stained by both female- and male-derived probes, unlike the Z chromosomes, which were preferentially stained by the male-derived probe in E. kuehniella only but were otherwise inconspicuous. The ratio of female:male staining and the pattern of staining along the W chromosomes was species specific. CGH shows that W chromosomes in these species are molecularly well differentiated from the Z chromosomes. The conspicuous binding of the male-derived probe to the W chromosomes is presumably due to an accumulation of common interspersed repetitive sequences. Received: 6 January 1999; in revised form: 28 January 1999 / Accepted: 11 February 1999     

Venturia canescens parasitizing Galleria mellonella and Anagasta kuehniella: differing suitability of two hosts with highly variable growth potential

Venturia canescens (Grav.) (Hymenoptera: Ichneumonidae) is a solitary larval koinobiont endoparasitoid, ovipositing in several larval instars of different pyralid moth species that are pests of stored food products. After oviposition, the host larva continues to feed and grow for at least several days, the precise time doing so depending on the stage attacked. We examined the relationship between host stage and body mass on parasitoid development in late second to fifth instars of two hosts with highly variable growth potential: the wax moth, Galleria mellonella (L) and the flour moth, Anagasta kuehniella (Zeller)(Lepidoptera: Pyralidae). G. mellonella is the largest known host of V. canescens, with healthy larvae occasionally exceeding 400mg at pupation, whereas those of A. kuehniella rarely exceed 40 mg at the same stage. Parasitoid survival was generally higher in early instars of G. mellonella than in later instars. By contrast, percentage adult emergence in A. kuehniella was highest in late fifth instar and lowest in late second instar. A. kuehniella was the more suitable host species, with over 45% adult emergence in all instars, whereas in G. mellonella we found less than 35% adult emergence in all instars. Adult parasitoid size increased and egg-to-adult development time decreased in a host size- and instar-specific manner from A. kuehniella. The relationship between host size and stage and these fitness correlates was less clear in G. mellonella. Although both host species were parasitized over a similar range of fresh weights, the suitability weight-range of A. kuehniella was considerably wider than G. mellonella for the successful development of V. canescens. However, in hosts of similar weight under 5 mg when parasitized, larger wasps emerged from G. mellonella than from A. kuehniella. Parasitoid growth and development is clearly affected by host species, and we argue that patterns of host utilization and resource acquisition by parasitoids have evolved in accordance with host growth potential and the nutritional requirements of the parasitoid.     

Codling moth cytogenetics: karyotype, chromosomal location of rDNA, and molecular differentiation of sex chromosomes.

We performed a detailed karyotype analysis in the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), the key pest of pome fruit in the temperate regions of the world. The codling moth karyotype consisted of 2n = 56 chromosomes of a holokinetic type. The chromosomes were classified into 5 groups according to their sizes: extra large (3 pairs), large (3 pairs), medium (15 pairs), small (5 pairs), and dot-like (2 pairs). In pachytene nuclei of both sexes, a curious NOR (nucleolar organizer region) bivalent was observed. It carried 2 nucleoli, each associated with one end of the bivalent. FISH with an 18S ribosomal DNA probe confirmed the presence of 2 clusters of rRNA genes at the opposite ends of the bivalent. In accordance with this finding, 2 homologous NOR chromosomes were identified in mitotic metaphase, each showing hybridization signals at both ends. In highly polyploid somatic nuclei, females showed a large heterochromatin body, the so-called sex chromatin or W chromatin. The heterochromatin body was absent in male nuclei, indicating a WZ/ZZ (female/male) sex chromosome system. In keeping with the sex chromatin status, pachytene oocytes showed a sex chromosome bivalent (WZ) that was easily discernible by its heterochromatic W thread. To study molecular differentiation of the sex chromosomes, we employed genomic in situ hybridization (GISH) and comparative genomic hybridization (CGH). GISH detected the W chromosome by strong binding of the Cy3-labelled, female-derived DNA probe. With CGH, both the Cy3-labelled female-derived probe and Fluor-X labelled male-derived probe evenly bound to the W chromosome. This suggested that the W chromosome is predominantly composed of repetitive DNA sequences occurring scattered in other chromosomes but accumulated in the W chromosome. The demonstrated ways of W chromosome identification will facilitate the development of genetic sexing strains desirable for pest control using the sterile insect technique.     

Correlation between fibroin amino acid sequence and physical silk properties

The fiber properties of lepidopteran silk depend on the amino acid repeats that interact during H-fibroin polymerization. The aim of our research was to relate repeat composition to insect biology and fiber strength. Representative regions of the H-fibroin genes were sequenced and analyzed in three pyralid species: wax moth (Galleria mellonella), European flour moth (Ephestia kuehniella), and Indian meal moth (Plodia interpunctella). The amino acid repeats are species-specific, evidently a diversification of an ancestral region of 43 residues, and include three types of regularly dispersed motifs: modifications of GSSAASAA sequence, stretches of tripeptides GXZ where X and Z represent bulky residues, and sequences similar to PVIVIEE. No concatenations of GX dipeptide or alanine, which are typical for Bombyx silkworms and Antheraea silk moths, respectively, were found. Despite different repeat structure, the silks of G. mellonella and E. kuehniella exhibit similar tensile strength as the Bombyx and Antheraea silks. We suggest that in these latter two species, variations in the repeat length obstruct repeat alignment, but sufficiently long stretches of iterated residues get superposed to interact. In the pyralid H-fibroins, interactions of the widely separated and diverse motifs depend on the precision of repeat matching; silk is strong in G. mellonella and E. kuehniella, with 2-3 types of long homogeneous repeats, and nearly 10 times weaker in P. interpunctella, with seven types of shorter erratic repeats. The high proportion of large amino acids in the H-fibroin of pyralids has probably evolved in connection with the spinning habit of caterpillars that live in protective silk tubes and spin continuously, enlarging the tubes on one end and partly devouring the other one. The silk serves as a depot of energetically rich and essential amino acids that may be scarce in the diet.     

Retrotransposable elements on the W chromosome of the silkworm, Bombyx mori

The sex chromosomes of the silkworm, Bombyxmori, are designated ZW(XY) for females and ZZ(XX) for males. The W chromosome of B. mori does not recombine with the Z chromosome and autosomes and no genes for morphological characters have been mapped to the W chromosome as yet. Furthermore, femaleness is determined by the presence of a single W chromosome, regardless of the number of autosomes or Z chromosomes. To understand these interesting features of the W chromosome, it is necessary to analyze the W chromosome at the molecular biology level. Initially to isolate DNA sequences specific for the W chromosome as randomly amplified polymorphic DNA (RAPD) markers, we compared the genomic DNAs between males and females by PCR with arbitrary 10-mer primers. To the present, we have identified 12 W-specific RAPD markers, and with the exception of one RAPD marker, all of the deduced amino acid sequences of these W-specific RAPD markers show similarity to previously reported amino acid sequences of retrotransposable elements from various organisms. After constructing a genomic DNA lambda phage library of B. mori we obtained two lambda phage clones, one containing the W-Kabuki RAPD sequence and one containing the W-Samurai RAPD sequence and found that these DNA sequences comprised nested structures of many retrotransposable elements. To further analyze the W chromosome, we obtained 14 W-specific bacterial artificial chromosome (BAC) clones from three BAC libraries and subjected these clones to shotgun sequencing. The resulting assembly of sequences did not produce a single contiguous sequence due to the presence of many retrotransposable elements. Therefore, we coupled PCR with shotgun sequencing. Through these analyses, we found that many long terminal repeat (LTR) and non-LTR retrotransposons, retroposons, DNA transposons and their derivatives, have accumulated on the W chromosome as strata. These results strongly indicate that retrotransposable elements are the main structural component of the W chromosome.     

The genetic screening of preimplantation embryos by comparative genomic hybridisation

Comparative genomic hybridization (CGH) is an indirect DNA-based test which allows for the accurate analysis of aneuploidy involving any of the 24 types of chromosomes present (22 autosomes and the X and Y sex chromosomes). Traditionally, embryos have been screened using fluorescence in situ hybridization (FISH)--a technique that was limited in the number of chromosomes able to be identified in any one sample. Early CGH reports on aneuploidy in preimplantation embryos showed that any of the 24 chromosomes could be involved and so FISH methods were going to be ineffective in screening out abnormal embryos. Our results from routine clinical application of array CGH in preimplantation genetic diagnosis (PGD) patients confirm previous reports on patterns of chromosomal contribution to aneuploidy. The pregnancy outcomes following embryo transfer also indicate that despite the requirement to freeze embryos, rates are encouraging, and successful ongoing pregnancies can be achieved.     

Synteny and chromosome evolution in the lepidoptera: evidence from mapping in Heliconius melpomene

The extent of conservation of synteny and gene order in the Lepidoptera has been investigated previously only by comparing a small subset of linkage groups between the moth Bombyx mori and the butterfly Heliconius melpomene. Here we report the mapping of 64 additional conserved genes in H. melpomene, which contributed 47 markers to a comparative framework of 72 orthologous loci spanning all 21 H. melpomene chromosomes and 27 of the 28 B. mori chromosomes. Comparison of the maps revealed conserved synteny across all chromosomes for the 72 loci, as well as evidence for six cases of chromosome fusion in the Heliconius lineage that contributed to the derived 21-chromosome karyotype. Comparisons of gene order on these fused chromosomes revealed two instances of colinearity between H. melpomene and B. mori, but also one instance of likely chromosomal rearrangement. B. mori is the first lepidopteran species to have its genome sequenced, and the finding that there is conserved synteny and gene order among Lepidoptera indicates that the genomic tools developed in B. mori will be broadly useful in other species.     

Evidence for an immune function of lepidopteran silk proteins

Hemolymph coagulation stops bleeding and protects against infection. Clotting factors include both proteins that are conserved during evolution as well as more divergent proteins in different species. Here we show that several silk proteins also appear in the clot of the greater wax moth Galleria mellonella. RT-PCR analysis reveals that silk proteins are expressed in immune tissues and induced upon wounding in both Galleria and Ephestia kuehniella, a second pyralid moth. Our results support the idea that silk proteins were co-opted for immunity and coagulation during evolution.     

Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization

Comparative genomic in situ hybridization (CGH) provides a new possibility for searching genomes for imbalanced genetic material. Labeled genomic test DNA, prepared from clinical or tumor specimens, is mixed with differently labeled control DNA prepared from cells with normal chromosome complements. The mixed probe is used for chromosomal in situ suppression (CISS) hybridization to normal metaphase spreads (CGH-metaphase spreads). Hybridized test and control DNA sequences are detected via different fluorochromes, e.g., fluorescein isothiocyanate (FITC) and tetraethylrhodamine isothiocyanate (TRITC). The ratios of FITC/TRITC fluorescence intensities for each chromosome or chromosome segment should then reflect its relative copy number in the test genome compared with the control genome, e.g., 0.5 for monosomies, 1 for disomies, 1.5 for trisomies, etc. Initially, model experiments were designed to test the accuracy of fluorescence ratio measurements on single chromosomes. DNAs from up to five human chromosome-specific plasmid libraries were labeled with biotin and digoxigenin in different hapten proportions. Probe mixtures were used for CISS hybridization to normal human metaphase spreads and detected with FITC and TRITC. An epifluorescence microscope equipped with a cooled charge coupled device (CCD) camera was used for image acquisition. Procedures for fluorescence ratio measurements were developed on the basis of commercial image analysis software. For hapten ratios 4/1, 1/1 and 1/4, fluorescence ratio values measured for individual chromosomes could be used as a single reliable parameter for chromosome identification. Our findings indicate (1) a tight correlation of fluorescence ratio values with hapten ratios, and (2) the potential of fluorescence ratio measurements for multiple color chromosome painting. Subsequently, genomic test DNAs, prepared from a patient with Down syndrome, from blood of a patient with Tcell prolymphocytic leukemia, and from cultured cells of a renal papillary carcinoma cell line, were applied in CGH experiments. As expected, significant differences in the fluorescence ratios could be measured for chromosome types present in different copy numbers in these test genomes, including a trisomy of chromosome 21, the smallest autosome of the human complement. In addition, chromosome material involved in partial gains and losses of the different tumors could be mapped to their normal chromosome counterparts in CGH-metaphase spreads. An alternative and simpler evaluation procedure based on visual inspection of CCD images of CGH-metaphase spreads also yielded consistent results from several independent observers. Pitfalls, methodological improvements, and potential applications of CGH analyses are discussed.     

Comparative Genomic Hybridization As a New Method for Detection of Genomic Imbalance

Comparative Genomic Hybridization (CGH) is a molecular cytogenetic method for detecting chromosomal imbalances by comparing the copy number of DNA sequences in cells of tested tissue and the reference specimen. CGH is based on two-color fluorescence suppressive in situ hybridization of genomic test and reference DNAs, each labeled with a different fluorochrome, to metaphase chromosomes of a healthy individual. First described by Kallioniemi et al. in 1992, the CGH assay has been widely used for identification and characterization of both numerical and unbalanced structural chromosome abnormalities in cells of different tissues at various pathological conditions in humans, especially in tumor diseases. We discuss the specific features and quality control of comparative genomic hybridization, its advantages and limitations in detection of genomic imbalance and the prospects for development of this technology.     

Comparative genomic hybridisation for the analysis of chromosomal imbalances in solid tumours and haematological malignancies

 Comparative genomic hybridisation (CGH) is based on a two-colour, competitive fluorescence in situ hybridisation of differentially labelled tumour and reference DNA to normal metaphase chromosomes. This new technology has made a great impact in molecular tumour pathology due to its possible application to archival specimens and the ability to create copy number karyotypes throughout the whole genome from very small amounts of DNA. If chromosomal imbalances can be correlated with a etiological and clinical features of tumours, CGH could be able to provide new prognostic and diagnostic criteria. CGH findings further provide starting points for the molecular genetic characterisation of altered chromosomal regions harbouring yet unidentified genes involved in tumorigenesis and tumour progression. An overview of the results of published CGH studies on solid tumours and haematological malignancies is presented. Methodological limitations of the CGH technology are reported, as well as future developments which will improve its use in routine analysis. Accepted: 29 July 1997     

High-resolution comparative hybridization to combed DNA fibers

Comparative genomic hybridization (CGH) has proven to be a comprehensive new tool to detect genetic imbalances in genomic DNA. However, the resolution of this method carried out on normal human metaphase spreads is limited to low copy number gains and losses of ≥ 10 Mb. An improved resolution allowing the detection of copy number representations of single genes would strongly enhance the applicability of CGH as a diagnostic and research tool. This goal may be achieved when metaphase chromosomes are replaced by an array of target DNAs representing the genes of interest. To explore the feasibility of such a development in a model system we used cosmid MA2B3, which encompasses about 35 kb in the vicinity of exon 48 of the human dystrophin gene. Linearized cosmid fibers were attached to a glass surface and aligned in parallel by “molecular combing”. Two-color fluorescence in situ suppression hybridization was performed on these cosmid fibers with probe mixtures containing different ratios (ranging from 1:2 to 4:1) of biotin- and digoxigenin-labeled MA2B3 cosmid DNAs. For each mixture fluorescence ratios were determined for 40–50 individual combed DNA molecules. In two series comprising a total of 651 molecules the median fluorescence ratio measurements revealed a linear relationship with the chosen probe ratios. Our study demonstrates that fluorescence ratio measurements on single DNA molecules can be performed successfully. Received: 5 August 1996 / Revised: 30 September 1996     

Sex chromosome pairing and sex chromatin bodies in W-Z translocation strains of Ephestia kuehniella (Lepidoptera).

Structure and pairing behavior of sex chromosomes in females of four T(W;Z) lines of the Mediterranean flour moth, Ephestia kuehniella, were investigated using light and electron microscopic techniques and compared with the wild type. In light microscopic preparations of pachytene oocytes of wild-type females, the WZ bivalent stands out by its heterochromatic W chromosome strand. In T(W;Z) females, the part of the Z chromosome that was translated onto the W chromosome was demonstrated as a distal segment of the neo-W chromosome, displaying a characteristic non-W chromosomal chromomere-interchromomere pattern. This segment is homologously paired with the corresponding part of a complete Z chromosome. In contrast with the single ball of heterochromatic W chromatin in highly polyploid somatic nuclei of wild-type females, the translocation causes the formation of deformed or fragmented W chromatin bodies, probably owing to opposing tendencies of the Z and W chromosomal parts of the neo-W. In electron microscopic preparations of microspread nuclei, sex chromosome bivalents were identified by the remnants of electron-dense heterochromatin tangles decorating the W chromosome axis, by the different lengths of the Z and W chromosome axes, and by incomplete pairing. No heterochromatin tangles were attached to the translocated segment of the Z chromosome at one end of the neo-W chromosome. Because of the homologous pairing between the translocation and the structurally normal Z chromosome, pairing affinity of sex chromosomes in T(W;Z) females is significantly improved. Specific differences observed among T(W;Z)1-4 translocations are probably due to the different lengths of the translocated segments.     

Comparative genomic hybridization as a new method for detection of genomic imbalance

Comparative Genomic Hybridization (CGH) is a molecular cytogenetic analysis that allows identification of genomic changes by comparing the copy number of DNA sequences in cells of tested tissue and the reference specimen. CGH is based on competitive suppressive in situ hybridization of two differently labeled DNA probes (tested and reference, karyotypically normal, fluorochrome-labeled DNAs) with metaphase chromosomes of a healthy subject. First described by Kallioniemi et al. in 1992, the CGH assay has been widely used for identification and characterization of both numerical and structural chromosome abnormalities in cells of different tissues at various pathological conditions in humans, especially in tumor diseases. We discuss the specific features and quality control of comparative genomic hybridization, its advantages and limitations in detection of genomic imbalance and the prospects for development of this technology.     

Cytogenetic analysis from DNA by comparative genomic hybridization

Comparative genomic hybridization (CGH) is a modified in situ hybridization technique which allows detection and mapping of DNA sequence copy differences between two genomes in a single experiment. In CGH analysis, two differentially labelled genomic DNA (study and reference) are co-hybridized to normal metaphase spreads. Chromosomal locations of copy number changes in the DNA segments of the study genome are revealed by a variable fluorescence intensity ratio along each target chromosome. Since its development, CGH has been applied mostly as a research tool in the field of cancer cytogenetics to identify genetic changes in many previously unknown regions. CGH may also have a role in clinical cytogenetics for detection and identification of unbalanced chromosomal abnormalities.     

Characterization of double minute chromosomes’ DNA content in a human high grade astrocytoma cell line by using comparative genomic hybridization and fluorescence in situ hybridization

The presence of double minute chromosomes (dmin) in cancer cells is known to be correlated with gene amplifications. In human high grade astrocytomas or glioblastomas, about 50% of cytogenetically characterized cases display dmin. G5 is a cell line which has been established from a human glioblastoma containing multiple dmin. In order to identify the DNA content of these dmin, three techniques were successively used: conventional cytogenetic analysis, comparative genomic hybridization (CGH), and fluorescent in situ hybridization (FISH). The karyotype of G5 cells showed numerical chromosome changes (hypertriploidy), several marker chromosomes, and multiple dmin. CGH experiments detected two strong DNA amplification areas located in 9p21-22 and 9p24, as well as an underrepresentation of chromosomes 6, 10, 11, 13, 14, and 18q. By using FISH with a chromosome 9-specific painting probe to metaphase chromosomes of the G5 cell line, dmin were shown to contain DNA sequences originating from chromosome 9. This study demonstrates the usefulness of a combination of classical karyotyping, CGH, and FISH to identify the chromosomal origin of amplified DNA sequences in dmin. Received: 30 October 1994 / Revised: 25 February 1996     

Differential expansion of highly repeated DNA sequences in the swine subgenomes

Differences in highly repeated DNA sequences among three swine breeds genomes were detected by means of whole‐comparative genomic hybridization (W‐CGH). The results showed that Duroc, Iberian and Landrace/Large White breeds share similar DNA sequences in their centromeric regions, but the number of copies of the highly repeated DNA sequences building the blocks of heterochromatin in the metacentric chromosomes is differentially expanded among them. That is not the case in the acrocentric subgenome where the chromosomes share similar sequence composition and number of copies among the three breeds in the centromeric regions. The highly repeated DNA sequences in the chromosome Y also displayed differences among the breeds studied. The reported results are discussed in the light of the possible evolutionary tendencies of these particular DNA sequences.     

Sex chromosome polymorphism and heterogametic males revealed by two cloned DNA probes in the ZW/ZZ fish Leporinus elongatus

In order to study the divergence of teleost sex chromosomes, subtractive cloning was carried out between genomic DNA of males and females of the rainbow trout (XX/XY) and of Leporinus elongatus (ZW/ZZ). Inserts cloned in a plasmid vector were individually tested on Southern blots of DNA of males and females for sex specificity. No sex-specific insert was obtained from trout, but two out of ten inserts cloned from L. elongatus showed sex-specific patterns in this species: one corresponds to a sequence present on both Z and W chromosomes, while the other is W specific. Sequences of these two inserts show neither clear homology with other known sequences, nor an open reading frame. They cross-hybridize with the genomic DNA of Leporinus friderici, but without sex-specific patterns. Twenty-four L. elongatus adults were sexed by gonadal observation, chromosomed examination and Southern hybridization with one or the other insert. Ten males and 11 females had chromosomes and hybridization patterns typical of their sex. One ZW female was recognized as a male with the W-specific probe. This was also the case for two unusual ZW males, one having a male hybridization pattern with the other probe. These three atypical individuals may result from single genetic exchanges between four regions of the Z and the W, giving rise to three atypical W chromosomes. Finding males with such atypical heterochromosomes in a female heterogametic species may indicate that a gradual transition occurs between the heterogametic systems.     

Detection of DNA copy number changes in human endometriosis by comparative genomic hybridization

Endometriosis is characterized by infertility and pelvic pain in 10-15% of women of reproductive age. The genetic events involved in endometriotic cell expansion remain in large part unknown. To identify genomic changes involved in development of this disease, we examined a panel of 18 selected endometriotic tissues by comparative genomic hybridization (CGH), a molecular cytogenetic method that allows screening of the entire genome for chromosomal gains and/or losses. The study was performed on native, nonamplified DNA extracted from manually dissected endometriotic lesions. Recurrent copy number losses on several chromosomes were detected in 15 of 18 cases. Loss of chromosome 1p and 22q were detected in 50% of the cases. Additional common losses occurred on chromosomes 5p (33%), 6q (27%), 7p(22%), 9q (22%), 16 (22%) as well as on 17q in one case. Gain of DNA sequences were seen at 6q, 7q and 17q in three cases. To validate the CGH data, selective dual-color FISH was performed using probes for the deleted regions on chromosomes 1, 7 and 22 in parallel with the corresponding centromeric probes. Cases showing deletion by CGH all had two signals at 1p36, 7p22.1 and 22q12 in less than 30% of the nuclei in comparison to the double centromeric labels found in more than 85% of the cells. These findings indicate that genes localized to previously undescribed chromosomal regions play a role in development and progression of endometriosis.