Mitogenome data of Hylobates abbotti Kloss, 1929 (Abbott's Gray Gibbon).

Abstract Hylobates moloch (Audebert, 1797), the Javan or silvery gibbon, is a pair-living small ape which is exclusively found in the western and central regions of the Indonesian island of Java. It represents the southernmost occurring species of the genus Hylobates and inhabits the canopy of tropical rainforests. It is foremost characterized by its long silvery-gray fur in combination with a lack of duet songs in mated pairs. Hylobates moloch is threatened by habitat loss as well as the illegal wildlife trade and is listed as “Endangered” (EN) by the IUCN Red List of Threatened Species.

Gibbons of the genus Hylobates, which inhabit Southeast Asia, show great diversity and comprise seven to nine species. Natural hybridisation has been observed in several species contact zones, but the history and extent of hybridisation and introgression in possibly historical and the current contact zones remain unclear. To uncover Hylobates species phylogeny and the extent of introgression in their evolution, genotyping by random amplicon sequencing-direct (GRAS-Di) was applied to 47 gibbons, representing seven Hylobates species/subspecies and two outgroup gibbon species. Over 200,000 autosomal single-nucleotide variant sites were identified. The autosomal phylogeny supported that divergence from the mainland species began ~3.5 million years ago, and subsequently occurred among the Sundaic island species. Significant introgression signals were detected between H. lar and H. pileatus, H. lar and H. agilis and H. albibarbis and H. muelleri, which all are parapatric and form ongoing hybrid zones. Furthermore, the introgression signals were detected in every analysed individual of these species, indicating a relatively long history of hybridisation, which might have affected the entire gene pool. By contrast, signals of introgression were either not detected or doubtful in other species pairs living on different islands, indicating the rarity of hybridisation and introgression, even though the Sundaic islands were connected during the Pliocene and Pleistocene glacial events.

Abstract. This research aims to determine the criminal liability against perpetrators of the protected animal sales of the Java OWA which has the scientific name Hylobates Moloch according to Law No. 5 of 1990 on the conservation of natural resources and its ecosystem and to know the effectiveness of criminal law against the animal sales of Java Owa protected. This method of research uses normative juridical use of secondary data in the form of primary, secondary, and tertiary legal materials obtained through libray research by using qualitative juridical specification which is a research that has the starting point of the legislation and is then analyzed qualitatively with the analysis of legal silogism by deducting. The results of research review of the trade of Owa Java animals related to Law No. 5 year 1990 on the conservation of natural resources and ecosystems has not provided protection to the animals of Owa Java because it proved to be field still many sales cases of Owa Java occurring throughout Indonesia, whether through conventional methods and online methods. Advice from this researcher so that the government is more attentive and caring about endemic animals such as the Javanese Owa to be not extinct. Then the government needs to revise the new conservation law with more complementary rules, as well as law enforcement officials must be active in addressing protected animal sales cases.

Semenogelin 1 and 2 (SEMG1 and SEMG2) are known as semen coagulating proteins in primates with a repetitive structure of 60-amino acids. The number of repeats varies among species and is hypothesized to be related to the level of primate sperm competition. Gibbons until recently were thought to be monogamous primates, but it is now known that gibbon social structure is flexible. Thus, hypotheses of the relationship between the SEMGs evolution and mating systems were tested. The sequences of the exon 2 of the SEMG1 and SEMG2 were obtained from 50 captive gibbons comprising six species belonging to three genera (Hylobates, Symphalangus, and Nomascus). Then we quantified the levels of polymorphism and estimated rates of protein evolution by calculating d N /d S ratio. Several mutations that create a premature stop codon in the SEMG1 and a reduction in the repeats of the SEMG2 in the genus Hylobates were observed and may alter the coding properties for these proteins. We also found different level of nucleotide diversity in each gene and between genera. Strikingly, in Nomascus leucogenys we discovered a high d N /d S ratio in the SEMG1 and SEMG2. The Nomascus SEMG2 also showed significantly lower nucleotide diversity than the other two genera. These results are consistent with the presence of a strong positive selection in the Nomascus lineage even if the exact selective forces acting on these genes are not yet conclusively known. We were not able to demonstrate, among gibbons, unambiguous relationships between the SEMGs evolution and mating systems.

The objective of this study was to reconstruct phylogenetic relationships of gibbons (3 genera, 11 species) deduced from complete sequenced mitochondrial genome sequences. According to conserved (C) / variable (V) sites ratio test, 9 selected protein coding genes were combined into a sequence with 9356 bases long including gaps. A resolved phylogenetic tree was obtained for the mitochondrial genome in the maximum-likelihood and maximum-parsimony analyses. In accordance with all previous morphological and molecular evidence, our results clearly supported monophyly of family Hylobatidae with predominantly strongly supported and each of the three genera with high support based on these coding genes. Among three genera, first Nomascus, next Symphalangus and at last Hylobates diverged, which identical to other previous research based on the whole mitochondrial genome. Our phylogenetic relationships of Nomascus group accord with Chan et al. (2010), only support values of a node of N. gabriellae (90%) were slight lower than Chan's result (97%). Among genus Hylobates, in our result, 6 species are involved and H. pileatus, H. lar, H. klossii, H. agilis, H. moloch and H. muelleri diverged sequentially.

Arsitektur Rigi-rigi Dermatograf dari Marga Hylobates dan Symphalangus (Famili HYLOBATIDAE) di Indonesia

Tesso Nilo area which is located at Riau province covers an area of 188.00 ha. Recently, it is famous because of a sharply increased in encroachment activities for forest conversion, especially for oil palm plantations and village sites. It was conducted in logged forest around Segati river, Toro river, Mamahan river and Sawan river in June 2003. The results showed that the area had the high richness of plant species which was indicated by the high value of Mennhenick index. Records from the 1 ha studied plot identified a total of 360 species included 165 genera and 57 families with 215 tree species 305 sapling species. Some important tree species which were included in the Red List of IUCN were ‘gaharu’ (Aquilaria malaccensis), ‘ramin’ (Gonystylus bancanus), Shorea spp. and Dipterocarpus spp. The local community utilized 83 species of medicinal plants and 4 species of toxic plants for fishing. The total number of recorded bird species was 114 species represented 29% of the total Sumatran bird species. The most important bird species were the Wrinkled Hornbill (Aceros corrugatus), Crestless Fireback (Lophura erythrophthalma), Crested Fireback (Lophura ignita) and Hooked-billed Bulbul (Setornis criniger). The total number of recorded mammal species was 34 species or 16.5% of 206 species of Sumatran mammals. The most important mammal species included Sumatran tiger (Panthera tigris sumatrae), Sumatran elephant (Elephas maximus sumatrensis), the Sun bear (Helarctos malayanus) and three species of primates: Hylobates agilis, Presbytis femoralis and Macaca nemestrina. The herpetofauna contained 15 reptile species and 18 amphibian species. The most important herpetofauna was the endangered False Gharial (Tomistoma schlegelii) and the vulnerable Common Softshelled turtle (Amyda cartilaginea). The number of recorded fish species was 50 represented 18% of the total Sumatran fish species (272 species). The important fish species were Breinsteinea sp. and Chaca bankanensis which were unique and rare. Since insects are the largest group of animal, this study only focused on beetles. The identified beetles were classified into 644 species and 34 families. The important beetles were the Longhorn beetles (Cerambycidae) and the Scarab beetles (Scarabaeidae).The small mammal parasites consisted of ectoparasites which were categorized into 14 species and endoparasites which were categorized into 2 orders and 3 species. Keywords: biodiversity, logged forest, richness, Sumatran tiger, Sumatran elephant

The feeding behavior and of hylobates syndactylus (siamang) was investigated at the Way-Canguk Research area inBukit-Barisan Selatan National Park between the months of February and July 2002. The research was conducted in undisturbedforest area (F1) and disturbed forest area (F2). Focal animal sampling method was use with and interval of 5 minutes. Sixsiamangs in each forest types were observed. The results of the investigations concluded that F1 and F2 siamangs spend moretime resting than feeding. The percentage of feeding activity whichy is done by F1 and F2 Siamangs are 31% and 34%. Theactivity began at 6 until 7 am and increased to midday until 2 pm. After that, the activity decreased until 5.30 pm. The monthlyrange correlates negatively activity of F1 and F2 siamangs use mostly the middle layer of the canopy.Keywords : The feeding activites, Hylobates syndactylus, Bukit -Barisan Selatan National Park, disturbed, undisturbed.

小型類人猿シロテテナガザル（Hylobates lar）について犬歯形態の詳細な記載と大きさの性的二型性を明らかにすることを目的に研究を行った。テナガザルの犬歯は従来いわれているように性的二型性が小さく，雌雄間で形態が非常によく似ている。歯冠頬側面の概形は上顎犬歯でサーベル形，下顎犬歯は不正四辺形を呈する。オスに較べてメスの形態特徴を挙げると，1）サイズが小さい，2）歯冠浮彫像の発達が弱く，全体に丸みを帯びている，3）下顎犬歯の近心shoulderの位置が歯冠高の約1/2にある，4）歯頚隆線がよく発達していることである。歯冠サイズによる犬歯の性差では，上・下顎の歯冠基底部のサイズや歯冠高でオスの方が有意に大きい。一方，下顎犬歯では歯冠近遠心径に対する歯頚部エナメル質の膨らみはメスの方が大きく，有意に強い性差を示す。歯冠の高径，とくに下顎犬歯の尖頭から近心shoulderまでの距離が最も強い性差を示す。犬歯の形態やサイズに性的二型がみられることはペア社会を構成するテナガザルでもある程度雌雄の違いが大きさや形にも存在することを示す。

Conservation of dwarf gibbons (Hylobates) and siamangs (Symphalangus): Status, challenges and opportunities Status, challenges and opportunities

BackgroundThe evolutionary relationships of closely related species have long been of interest to biologists since these species experienced different evolutionary processes in a relatively short period of time. Comparison of phylogenies inferred from DNA sequences with differing inheritance patterns, such as mitochondrial, autosomal, and X and Y chromosomal loci, can provide more comprehensive inferences of the evolutionary histories of species. Gibbons, especially the genus Hylobates, are particularly intriguing as they consist of multiple closely related species which emerged rapidly and live in close geographic proximity. Our current understanding of relationships among Hylobates species is largely based on data from the maternally-inherited mitochondrial DNAs (mtDNAs).ResultsTo infer the paternal histories of gibbon taxa, we sequenced multiple Y chromosomal loci from 26 gibbons representing 10 species. As expected, we find levels of sequence variation some five times lower than observed for the mitochondrial genome (mtgenome). Although our Y chromosome phylogenetic tree shows relatively low resolution compared to the mtgenome tree, our results are consistent with the monophyly of gibbon genera suggested by the mtgenome tree. In a comparison of the molecular dating of divergences and on the branching patterns of phylogeny trees between mtgenome and Y chromosome data, we found: 1) the inferred divergence estimates were more recent for the Y chromosome than for the mtgenome, 2) the species H. lar and H. pileatus are monophyletic in the mtgenome phylogeny, respectively, but a H. pileatus individual falls into the H. lar Y chromosome clade.ConclusionsBased on the ~6.4 kb of Y chromosomal DNA sequence data generated for each of the 26 individuals in this study, we provide molecular inferences on gibbon and particularly on Hylobates evolution complementary to those from mtDNA data. Overall, our results illustrate the utility of comparative studies of loci with different inheritance patterns for investigating potential sex specific processes on the evolutionary histories of closely related taxa, and emphasize the need for further sampling of gibbons of known provenance.

Compared with the great apes, the small-bodied hylobatids were treated historically as a relatively uniform group with 2 genera, Hylobates and the larger-bodied Symphalangus. Four genera are now recognized, each with a different chromosome number: Hoolock (hoolock) (38), Hylobates (44), Nomascus (crested gibbon) (52), and Symphalangus (siamang) (50). Previous morphological studies based on relative bone lengths, e.g., intermembral indices; molar tooth sizes; and body masses did not distinguish the 4 genera from each other. We applied quantitative anatomical methods to test the hypothesis that each genus can be differentiated from the others using the relative distribution of body mass to the forelimbs and hind limbs. Based on dissections of 13 hylobatids from captive facilities, our findings demonstrate that each of the 4 genera has a distinct pattern of body mass distribution. For example, the adult Hoolock has limb proportions of nearly equal mass, a pattern that differentiates it from species in the genus Hylobates, e.g., H. lar (lar gibbon), H. moloch (Javan gibbon), H. pileatus (pileated gibbon), Nomascus, and Symphalangus. Hylobates is distinct in having heavy hind limbs. Although Symphalangus has been treated as a scaled up version of Hylobates, its forelimb exceeds its hind limb mass, an unusual primate pattern otherwise found only in orangutans. This research provides new information on whole body anatomy and adds to the genetic, ecological, and behavioral evidence for clarifying the taxonomy of the hylobatids. The research also underscores the important contribution of studies on rare species in captivity.

BackgroundGibbons or small apes inhabit tropical and subtropical rain forests in Southeast Asia and adjacent regions, and are, next to great apes, our closest living relatives. With up to 16 species, gibbons form the most diverse group of living hominoids, but the number of taxa, their phylogenetic relationships and their phylogeography is controversial. To further the discussion of these issues we analyzed the complete mitochondrial cytochrome b gene from 85 individuals representing all gibbon species, including most subspecies.ResultsBased on phylogenetic tree reconstructions, several monophyletic clades were detected, corresponding to genera, species and subspecies. A significantly supported branching pattern was obtained for members of the genus Nomascus but not for the genus Hylobates. The phylogenetic relationships among the four genera were also not well resolved. Nevertheless, the new data permitted the estimation of divergence ages for all taxa for the first time and showed that most lineages emerged during four short time periods. In the first, between ~6.7 and ~8.3 mya, the four gibbon genera diverged from each other. In the second (~3.0 - ~3.9 mya) and in the third period (~1.3 - ~1.8 mya), Hylobates and Hoolock differentiated. Finally, between ~0.5 and ~1.1 mya, Hylobates lar diverged into subspecies. In contrast, differentiation of Nomascus into species and subspecies was a continuous and prolonged process lasting from ~4.2 until ~0.4 mya.ConclusionsAlthough relationships among gibbon taxa on various levels remain unresolved, the present study provides a more complete view of the evolutionary and biogeographic history of the hylobatid family, and a more solid genetic basis for the taxonomic classification of the surviving taxa. We also show that mtDNA constitutes a useful marker for the accurate identification of individual gibbons, a tool which is urgently required to locate hunting hotspots and select individuals for captive breeding programs. Further studies including nuclear sequence data are necessary to completely understand the phylogeny and phylogeography of gibbons.

Ovarian cycles were determined for two captive females of the yellow-cheeked crested gibbon (Nomascus gabriellae) using urinary sex steroids. The mean cycle length was 21.1±1.2 days (n = 7 cycles). The interval between any peak in oestrone concentration and the corresponding oestradiol peak had a range of 0-1 days, and cycle lengths determined with oestrone differed from those determined with oestradiol by 0-2 days. Neither hormone tended to peak earlier than the other. In female 1, menarche probably occurred just before or around the beginning of the colour transition from the black juvenile to the adult yellow fur coloration, whereas the older female 2 apparently began to exhibit regular cycles during this study, years after changing to adult fur colouration. Mean cycle lengths determined in this study for N. gabriellae were virtually identical to those for those of other gibbons determined in previous studies applying endocrinological methods (Hylobates spp.: 20.0-25.4 days, N. leucogenys: 21.9, Symphalangus syndactylus: 21.8). These values are, in most cases, similar to intervals determined between peaks of sexual swellings. On the other hand, published cycle lengths based on intervals between menstrual bleedings or between copulations tend to be considerably longer. Because some cycles may easily remain undetected with the latter two methods, the resulting intervals may not be reliable indicators of the duration of menstrual cycles in gibbons. Cycles of gibbons appear to be shorter than those of other primates, apart from some – but not all – New World monkeys.

A longevity record of 60 years spent in captivity by a Mueller’s gibbon (Hylobates muelleri) is reported here. This appears to be the second-highest age so far reported for a non-human primate, but it is especially remarkable when adjusted for body size. It is well known that longevity in mammals correlates with body weight. Small apes should, therefore, be expected to exhibit lower longevity than the great apes because of their lower body weight. However, the longevity record for Hylobates reverses this expectation for great apes like orangutans (Pongo) and gorillas (Gorilla). This study further found a significant correlation between the captive population size of primate genera and their recorded longevity. A comparison of longevity and captive population size suggests that recorded longevity in the gibbon genera Hoolock, Nomascus and Symphalangus is lower than that of the genus Hylobates because Hylobates is kept in captivity in much higher numbers. As a result, data on Hylobates longevity are obtained from larger sample sizes than that of all other gibbons. This suggests that all gibbon genera may eventually be revealed to exhibit an elevated longevity in relation to their body weight when larger amounts of data become available. Longevity data for great apes, in contrast, are based on larger samples than those for most genera of the small apes, and an increase in sample size for great ape genera may less likely produce a substantial increase in the longevity record.

In this study we characterized the extension, reciprocal arrangement, and orientation of syntenic chromosomal segments in the lar gibbon (Hylobates lar, HLA) by hybridization of a panel of approximately 1000 human BAC clones. Each lar gibbon rearrangement was defined by a splitting BAC clone or by two overlapping clones flanking the breakpoint. A reconstruction of the synteny arrangement of the last common ancestor of all living lesser apes was made by combining these data with previous results in Nomascus leucogenys, Hoolock hoolock, and Symphalangus syndactylus. The definition of the ancestral synteny organization facilitated tracking the cascade of chromosomal changes from the Hominoidea ancestor to the present day karyotype of Hylobates and Nomascus. Each chromosomal rearrangement could be placed within an approximate phylogenetic and temporal framework. We identified 12 lar-specific rearrangements and five previously undescribed rearrangements that occurred in the Hylobatidae ancestor. The majority of the chromosomal differences between lar gibbons and humans are due to rearrangements that occurred in the Hylobatidae ancestor (38 events), consistent with the hypothesis that the genus Hylobates is the most recently evolved lesser ape genus. The rates of rearrangements in gibbons are 10 to 20 times higher than the mammalian default rate. Segmental duplication may be a driving force in gibbon chromosome evolution, because a consistent number of rearrangements involves pericentromeric regions (10 events) and centromere inactivation (seven events). Both phenomena can be reasonably supposed to have strongly contributed to the euchromatic dispersal of segmental duplications typical of pericentromeric regions. This hypothesis can be more fully tested when the sequence of this gibbon species becomes available. The detailed synteny map provided here will, in turn, substantially facilitate sequence assembly efforts.

The course of chromosome evolution in small apes is still not clear, though painting analyses have opened the way for elucidating the puzzle. Even the C-banding pattern of the lar-group of gibbons (the genus Hylobates) is not clarified yet, although our previous studies suggested that lar-group gibbons have a unique C-banding pattern. We therefore made observations to establish C-banded karyotypes of the agile gibbons included in the lar-group. The data were compared with those of siamangs (the genus Symphalangus), which carry distinctive C-bands, to determine the chromosomal patterns in each group. C-banded chromosomes of agile gibbons showed several terminal, interstitial and paracentric bands, whose patterns are specific for each chromosome, whereas the C-bands of siamangs were located only at the terminal and centromeric regions in most chromosomes. Moreover, the C-bands of agile gibbons and siamangs were shown to be G+C-rich and A+T-rich DNA, respectively, by DAPI/C-band sequential staining. Additionally, PRINS labelling with a telomere primer revealed that agile gibbons have telomeric DNA only at chromosome ends where there is no C-band (non-telomeric heterochromatin), whereas the telomeric DNA of siamangs is located in the terminal C-banded regions (telomeric heterochromatin). Although the evolutionary mechanisms in small apes are still unknown, C-banding patterns and distribution of telomeric DNA sequences should provide valuable data to deduce the evolutionary pathways of small apes.

Social organization involving pair bonding and two-adult groups is rare in mammals. Current sociobiological theory suggests that this grouping and behavior pattern is somewhat anomalous. The gibbons (genus Hylobates) are the only hominoids to exhibit pair bonds and two-adult groups. In this article I present an overview of the current issues in monogamy and pair-bond theory, and review traditional conceptualizations and the accumulated data relevant to gibbon social organization. The significance of hominoid behavioral phylogeny and population-wide studies is also considered. Recent findings indicate that pair-bonding and two-adult groups are not ubiquitous among the hylobatids. Many aspects of gibbon behavior and ecology do not conform to expectations of the conditions under which two-adult groups and/or pair-bonding patterns should evolve. A review of the information available from long-term and short-term studies of gibbons suggests an alternative way of viewing their socioecology. I propose that gibbons currently exist in variable communities that have arisen via ecological pressures and specific behavioral patterns from an ancestral multimale/multi-female grouping pattern. This social organization is not best characterized as "monogamous." This review also suggests that hominoid grouping patterns can be viewed as occurring along a continuum rather than as being discretely different units.

The role of cytogenetic analysis in the diagnosis and management of haematological malignancies is undisputed.The accuracy of cytogenetic diagnosis has improved steadily over the past 20 years, primarily due to a series of technical developments. However, despite improvements in high-resolution banding and culture methods to detect the chromosomally abnormal cells, many haematological malignancies are retractable to conventional cytogenetic analysis. This may be due to the presence of multiple abnormal clones, complex rearrangements, a low mitotic index, or poor chromosome morphology. Since the late 1980s a range of techniques based around fluorescence in situ hybridization (FISH) have greatly enhanced cytogenetic analysis. These use a variety of nucleic acid sequences as probes to cellular DNA targets and serve to bridge the gap between molecular genetic and conventional cytogenetic methods. Virtually any genomic DNA can now be used as a probe with which to investigate a wide variety of DNA targets, from metaphase chromosomes to mechanically stretched DNA fibres. The simultaneous detection of multiple target regions is also possible, using differentially labelled probes detected by different colours. In research, FISH has played a pivotal role in the identification of non-random translocations and deletions, pinpointing regions which contain genes involved in leukaemogenesis. Now, at the cutting edge, a new set of resources and technical innovations herald a new era for molecular cytogenetics, with colour karyotyping, comparative genomic hybridization (CGH) microarrays and mutation detection using padlock probes providing the promise of the future. The number of applications for FISH is almost unlimited (see Table I for some pertinent examples). This review will concentrate on the most recent developments in FISH which have had a considerable impact on the cytogenetic diagnosis and study of haematological malignancies, with some insight into the possible future roles for this flexible technology. The application of FISH to metaphase chromosomes provides unequivocal evidence of chromosome rearrangements. There are many different types of cloned or uncloned DNA which can be used for as a probe for FISH (reviewed in Buckle & Kearney, 1994). However, the most commonly used probes in cytogenetic analysis of haematological malignancy are: (i) repetitive sequence centromeric probes, (ii) whole chromosome paints, and (iii) locus-specific probes. Chromosome-specific centromeric probes which target tandemly repeated alpha (or beta) satellite sequences present in the heterochromatin of the chromosome centromeres are used to detect numerical chromosome abnormalities. Centromeric probes are commercially available for all human chromosomes and these provide a rapid and simple way of enumerating specific chromosome pairs, both in metaphase and interphase. This type of analysis is useful in many types of leukaemia where the chromosome morphology is poor and banding indistinct, such as in hyperdiploid acute lymphoblastic leukaemia (ALL). However, centromeric probes only give information on the number of centromeres of a particular type present; they cannot tell whether the chromosome is structurally abnormal. Whole chromosome painting probes are complex mixtures of sequences from the entire length of a specific chromosome. These are also available for all human chromosomes, and can be used to delineate chromosome pairs (Cremer et al, 1988; Pinkel et al, 1988). Whole chromosome painting probes (paints) can be derived from chromosome-specific libraries, PCR amplification of flow-sorted chromosome fractions, or microdissected DNA specific for each chromosome (Collins et al, 1991; Telenius et al, 1992; Vooijs et al, 1993; Guan et al, 1996). Chromosome paints are most useful for identifying the components of highly rearranged and marker chromosomes, where the banding pattern cannot be relied upon. However, their usefulness is limited to metaphase analysis, as the extended chromosome domains in interphase are often diffuse and difficult to quantitate. In addition, chromosome painting is a relatively insensitive technique and cannot detect small interstitial deletions, duplications or inversions. The resolution for the detection of small telomeric translocations is also limited. Single locus probes detect specific sequences present in only one copy. When using these probes the efficiency of hybridization needs to be considered; the larger the target sequence the more efficient the hybridization. Single-copy probes cloned in cosmid, YAC, P1, PAC and BAC vectors all give reliable FISH signals, with a fluorescent signal on both chromosome homologues in >90% of metaphases. Structural rearrangements detected using this type of probe include translocations, inversions and specific deletions (Dauwerse et al, 1990; Tkachuk et al, 1992; Sacchi et al, 1995; Jaju et al, 1998). The use of specific gene probes for chromosomal translocations has simplified the process of identifying known translocations, especially in complex or masked versions of the translocation (e.g. BCR/ABL, PML/RARα fusions), and has particular applications for interphase analysis. One of the greatest advances in cytogenetic analysis facilitated by FISH has been the ability to use non-dividing cells as DNA targets, referred to as interphase FISH (Cremer et al, 1986). This enables the screening of large numbers of cells and provides access to a variety of sources of haemopoietic cells including blood and bone marrow smears and haemopoietic progenitor cells from colony assays (Bentz et al, 1993; Poddighe et al, 1993; Mühlmann et al, 1998). This has considerable advantages for some haemopoietic malignancies, where the proliferative activity is low, or when the mitotic cells do not represent the neoplastic clone, for example chronic lymphoblastic leukaemia (CLL), Hodgkin's disease, multiple myeloma. Interphase FISH permits the identification of both numerical and structural chromosome abnormalities both as an aid to cyto-genetic diagnosis and for monitoring disease progression. Interphase FISH has had a major impact on the cytogenetic analysis of B-CLL, revealing a much higher incidence of trisomy 12 than found by conventional cytogenetic analysis (Anastasi et al, 1992; Garcia-Marco et al, 1997). An examination of the relationship between clinical stage and trisomy 12 showed an association with atypical morphology, advanced stage of disease and low proliferative activity. In addition, immunophenotyping and FISH showed that the +12 is present in only a proportion of clonal B cells (Garcia-Marco et al, 1997). All of this data suggests that trisomy 12 is a secondary event in the development of CLL. For chromosome deletions, specific locus or region-specific probes have been used to demonstrate a high frequency of mono-allelic deletions of the RB1 and p53 genes in B-cell malignancies (Stilgenbauer et al, 1993, 1995; Döhner et al, 1995; Cano et al, 1996). Interphase FISH was also instrumental in identifying the critical region of deletion on 11q13 associated with B-cell lymphoid malignancy, which consequently identified mutations of the ATM gene in T-prolymphocytic leukaemia (PLL) (Stilgenbauer et al, 1997). DNA probes for the fusion genes involved most specific chromosomal translocations and inversions in leukaemia are now commercially available. The differential labelling and detection of these probes in different colours enables a direct visualization of the fusion gene. The simplest scheme is to use two probes (one from each of the fusion genes), differentially labelled and detected with two different-coloured fluorochromes (see Fig 1A). An interphase cell positive for the translocation will exhibit a red–green fusion signal representing the translocation, and a single red and green signal corresponding to the normal chromosome homologues. However, the false positive rate using this approach is quite high (approximately 5%). In addition, the presence of variant translocations or translocations in which the breakpoints are spread over a large distance (e.g. Burkitt's lymphoma), means that the false negative rate can also be quite high. There are several more complex strategies to overcome this (see Figs 1B and 1C). Firstly, if a series of probes spanning both translocation breakpoints are used, this will result in splitting of both fluorescent signals, and the presence of two red–green fusions. Another, more complex, strategy is to employ three or even four different colours, so that the incidence of false positives and false negatives is reduced (Ried et al, 1993; Sinclair et al, 1997). However, the more complicated the colour scheme, the more difficult and complex the analysis. At present, this analysis is done manually, so this is a serious consideration. . Schematic representation of the detection, by FISH, of the Philadelphia translocation in interphase nuclei. In each case the left-hand panel shows the location of the FISH signals on metaphase chromosomes (partial karyotype), and the right-hand panel the interphase FISH signals. In (A) two probes from the flanking regions of the BCR and ABL genes are labelled and detected in different colours: BCR in red and ABL in green. The BCR/ABL fusion results in co-localization of the red and green signals on the der(22) (Philadelphia) chromosome, with a single red and green signal separated, corresponding to the normal chromosomes 22 and 9, respectively. A BCR/ABL negative cell would show two separate red and two green signals. The scheme in (B) uses two probes, this time spanning both the BCR and ABL breakpoint regions. In this case, two red/green fusion signals are formed: one corresponding to the der(9), and the second to the der(22). A positive cell would therefore exhibit one red, one green and two red/green fusions (from Dewald et al, 1998). In (C), a third probe from the region just proximal to ABL on 9q34 is used, labelled in a different colour (represented here in yellow). A translocation positive cell exhibits one green/yellow doublet, one red/green and a single red and yellow signal (from Sinclair et al, 1997). The possibility of using interphase FISH as screening test for specific abnormalities found in acute myeloid leukaemia (AML) subtypes was recently described by Fischer et al (1996). This study used 23 different probes and six to eight hybridizations per patient. They found that interphase FISH was more sensitive for the detection of t(8;21), inv(16), +8q, +11q, +21q, +22q and −Y, and obtained a cytogenetic result in a proportion of cases with no evaluable metaphases. However, this kind of analysis may eventually be replaced by disease-specific DNA chips (see Matrix-CGH below). The detection of residual Philadelphia-positive cells is important after allogeneic bone marrow transplant or interferon (IFN) treatment. In particular, the degree of response to IFN treatment has been shown to be an independent prognostic indicator. The sensitivity of conventional cytogenetics is around 5%, and may be difficult due to low mitotic rate of cells after treatment. RT-PCR is the most sensitive method for detection of BCR-ABL (approximately 10−6) but quantification is difficult. Interphase FISH offers the prospect of using peripheral blood samples, reducing the need for frequent bone marrow aspirates. However, 'in house' cut-off levels must be established for each probe set. Conventional FISH probes for the detection of BCR-ABL gene fusion in interphase cells have suffered from a high false positive rate (Tkachuk et al, 1992). The development of three-colour/three-probe FISH protocols for BCR-ABL detection has significantly lowered the false positive rate, and also increased the sensitivity of detection (Sinclair et al, 1997; Dewald et al, 1998). Sinclair et al (1997) used a third probe (for the ASS gene) 200 kb proximal to ABL, such that when a true BCR-ABL fusion was present, there was one co-localization for BCR-ABL, and a separate ASS signal corresponding to the der(9). In cells where the BCR and ABL signals co-localized due to chance, the ASS signal co-localized with the red ABL signal on both chromosomes 9 (see Fig 1C). This three-colour approach resulted in a low false positive and false negative rate. Dewald et al (1998) used a similar strategy, with probes spanning both the BCR and ABL breakpoints. This resulted in two different co-localizations: one representing the der(22) and the other the der(9) chromosomes (see Fig 1B). Strict scoring criteria, experienced operators and scoring of >3000 cells all enabled the detection of residual disease in 0.079% of cells. This skilled and time-consuming approach was also successful in detecting variant translocations. Although the sensitivity of dual-colour interphase FISH is less than for RT-PCR, PCR is not a possibility in a number of cases, for example for the detection of deletions, monosomy or trisomy. Interphase FISH has been used for the detection of residual disease after allogeneic bone marrow transplantation (Anastasi et al, 1991; Wessman et al, 1993). Kasprzyk & Secker-Walker (1997) studied hyperdiploid karyotypes in ALL to detect minimal residual disease. Using three-colour interphase FISH, targeting three chromosomes simultaneously, they were able to achieve a sensitivity of 10−4, and predict relapse in a number of cases. The ability to combine interphase FISH analysis with immunological staining for cell surface antigens provides a powerful method to combine cell by cell analysis with morphology or immunophenotype. Simultaneous immunophenotyping and FISH analysis has been used to investigate lineage involvement in myelodysplastic syndrome (MDS), chronic myeloid leukaemia (CML) and other myeloproliferative syndromes (Price et al, 1992; Nylund et al, 1993; Torlakovic et al, 1994; Soenen et al, 1995; Haferlach et al, 1997, reviewed in Knuutila, 1997). Concurrent immunophenotype and FISH analysis has also been used to demonstrate that the leukaemia which emerged 5 years after sex-mismatched allogeneic bone marrow transplant occurred in donor cells (Katz et al, 1993). In CML, three-colour detection of the Philadelphia translocation and immunophenotype enabled the identification of the translocation in CD20-positive B cells (Torlakovic et al, 1994) and more recently CD3-positive T cells and CD34-positive precursor cells (Haferlach et al, 1997). This supports the belief that CML is a disorder of an early progenitor cell, capable of differentiating into myeloid and some lymphoid lineages (reviewed in Knuutila, 1997). There are also reports of the clonal involvement of B cells in MDS, using del(20q) and monosomy 7 as clonal markers (White et al, 1994; van Lom et al, 1995). In Hodgkin's disease the low percentage of Hodgkin and Reed-Sternberg (HRS) cells means that even interphase FISH may not detect clonal abnormalities. In a recent study the combination of CD30+ staining and FISH with pairs of centromeric probes revealed numerical abnormalities in 100% of HRS cells (Weber-Matthiesen et al, 1995). Surprisingly, clonal abnormalities found in metaphase analysis were not consistent with the interphase FISH analysis, indicating that metaphase analysis of Hodgkin's disease may not be informative. FISH has proved an invaluable aid in the mapping of translocation breakpoints, resulting in the identification of many fusion genes (reviewed in Rabbitts, 1994). A recent addition to the repertoire of FISH techniques now provides significant advantages over other molecular methods for mapping breakpoints which are dispersed over large distances. The term Fibre-FISH is used to describe a collection of methods for performing FISH to extended DNA stretched out on a glass slide (Wiegant et al, 1992; Parra & Windle, 1993; Bensimon et al, 1994; reviewed in Raap, 1998). vandraager et al (1996) have demonstrated the usefulness of this technique for mapping breakpoints of the cyclin D1 gene in mantle cell lymphomas. Using a series of overlapping probes from the 11q13 breakpoint region labelled in alternating red and green fluorochromes creates a colour bar code for the region. Translocations are recognized by the disruption of this bar code into its two complementary parts. The advantages of this method over Southern blotting or pulsed field gel electrophoresis are its simplicity and speed: only a few images need to be examined, and chromosomal breaks over a distance of 250 kb can be visualized. However, the parameters underlying the technique are poorly understood, and at present it remains a research rather than diagnostic tool, confined to a few specialist laboratories. The strength of conventional (G-banded) cytogenetic analysis has always been the ability to survey the entire genome for clues to pathogenesis. However, the poor chromosome morphology and low mitotic index of many leukaemias and lymphomas means that conventional cytogenetic analysis is often limited. In addition, the analysis of banding pattern in highly rearranged karyotypes is difficult and unreliable. One of the remaining challenges for the new FISH techniques is to identify cryptic rearrangements, particularly involving telomeric regions, in apparently normal karyotypes. A significant proportion (15–20%) of bone marrow karyotypes in leukaemia are reported as normal by conventional (G-banded) cytogenetic analysis. Despite significant improvements in the quality of leukaemic metaphase preparations over the past decade, the abnormality rate has not improved. The t(12;21)(p13;q22) remained undetected until 1994, despite the fact that it accounts for 25% of childhood B-cell ALL cases (Romana et al, 1994). This translocation still remains undetectable by conventional cytogenetic analysis. The difficulty in detecting chromosome abnormalities such as this in the fact that there is a of staining regions of a similar The recent development of whole chromosome painting provides the promise of identifying cryptic chromosome rearrangements, a of all chromosome abnormalities in a single FISH using the method of probe labelling was described by et al In this probes are labelled with mixtures of fluorochromes such that no two probes have the The number of targets which can be in this is where number of fluorochromes available. FISH with to different colours has been available for a number of years, using probes labelled with three fluorochromes (Dauwerse et al, 1992; et al, 1992). the number of fluorochromes to enables the identification of all pairs of human The has been due in to the of new fluorochromes in the and and to two detection methods to mixtures of fluorochromes et al, et al, 1996). of these used a set of whole chromosome paints, labelled with different mixtures of The detection FISH relied on separate images for each of using et al, 1996). The labelling combination for each chromosome was and in using The second used a single of the and a combination of and et al, 1996). An was used to the at each of the of these techniques have demonstrated chromosome rearrangements in complex karyotypes in cell and in haematological malignancies et al, et al, 1997; also Fig However, the sensitivity of both or remains to be The of this are the on metaphase analysis, and the resolution of painting probes. All of the available whole chromosome paints are in some of the particularly the telomeric regions. that the sensitivity of painting for the detection of translocations involving regions may be as low as also Fig In addition, whole chromosome painting will not detect deletions, duplications or inversions. In both and still to the and a combination of FISH is still to identify all abnormalities in complex karyotypes. . FISH to the analysis of a complex in the myeloid cell (A) after analysis. The structural abnormalities identified are: A cryptic was also present, but difficult to identify by analysis. (B) A metaphase after analysis. of amplification are in green and deletions in of the genome which are regions were identified the entire chromosome of and A deletion of due to the of an The analysis identified the of a marker chromosome, as as revealing several cryptic translocations in The of the analysis was to the of the with large deletions translocations in most cases. et al (1997) have recently described an approach which use of the regions of between different to approach a of colour of The of a colour for each chromosome was described by et al using a series of from the length of the chromosome, labelled differentially and detected in a different banding on the of between and has been useful in comparative of regions (reviewed in et al, 1997). The by and have now a set of paints derived from cell Chromosome-specific painting probes were derived from and by chromosome and When used for FISH to human metaphase chromosomes, this resulted in the of each chromosome into between two and six labelling using three fluorochromes resulted in a colour banding pattern for each chromosome. In with specific an colour can be Although at present the number of colour is the of this approach is with the to identify chromosomal inversions and colour banding has been used to identify cryptic translocations in CML A set of chromosome-specific probes which identify the of all human chromosomes the of the is now available for FISH of and of 1996). These contain DNA sequences cloned in cosmid, and PAC clones, the of which have been to between and kb from human chromosome These probes have been in a FISH for rearrangements on a series of with cryptic chromosome rearrangements et al, 1997). This is dual-colour an of all chromosome regions on a single However, the approach a high mitotic index and is most for the analysis of which on peripheral blood or where a cell is available. In the of these probes for leukaemic karyotypes has been to identify the specific region in rearrangements found by painting (see Fig An a FISH would the of all chromosome regions in a single However, the of labelling and multiple colour detection methods for cosmid, or even and PAC a series of developments. Firstly, the simultaneous analysis of all chromosome in a different colour the number of targets with fluorochromes is the of such an would on it is not whether the targets of such small probes labelled with several different fluorochromes can be due to of resolution of the is that the development of fluorochromes will this type of analysis . The use of chromosome-specific probes to identify the of chromosome on two In each case dual-colour hybridization was out with the probe labelled in and detected in red fluorescent and the probe labelled with and detected with fluorescent In (A) probes for and identified the on the as derived from In (B) the on was identified as from Although these abnormalities were detected by painting no information of the chromosomal region is also important to that the abnormality in (A) was described by as The of all of the new is that they still metaphase The advantages of are that it the need for cells and not any of the chromosome The is genomic and DNA are labelled with different in and normal metaphase The in number between the normal and is by in red and green fluorescence the length of the chromosome (see Fig the 5 years its et al, has a of identifying new regions of amplification and deletion in a wide variety of types (reviewed in et al, 1997). The use of for haematological malignancies is more limited (Bentz et al, et al, et al, et al, 1997). The of for haematological malignancies are the to detect rearrangements, and the for cells with the clonal However, and some lymphomas have from the application of (Bentz et al, et al, et al, 1996). One study of identified and not identified or not detected by clonal were identified in six out of cases with a normal (Bentz et al, The for results between and were a complex the or a of metaphases. This study that banding analysis may abnormalities and may important chromosome have not been identified in CLL. In a study of myeloid leukaemias found a between and results (Bentz et al, The only were a of to detect and The major of is its due to the on metaphase For deletions, the resolution of has been at (Bentz et al, 1998). the most future for in to cloned DNA (see below). This to overcome the of using metaphase chromosomes as a target for by metaphase chromosomes with cloned DNA in small and to the surface of a glass et al (1997) used for the detection of high number amplification using as For low number larger cloned probes or were For deletions, a resolution of it not between and The other of metaphase also at of clonal cells and will not detect translocations. One types of (i) disease-specific probe (ii) or (iii) DNA for specific regions, at over the whole probes are DNA in which the has been replaced by The rapid and of with complementary DNA sequences for a number of including FISH probes have been for the human telomeric the fluorescent detection of all in a single These signals to be a for fluorescence than conventional FISH signals, an of length et al, 1996). This may also be extended to other sequences such as centromeric may also be to combine telomeric probes with the chromosome-specific DNA probes to provide a of and specific chromosome This new the promise of detecting single in cells. probes of two different each 20 by a When the probe sequence the the and of the probe are and the probe is et al (1997) used two different probes, each labelled with a different to detect single in a centromeric The sensitivity of this may be improved by the of new sensitive labelling techniques such as the use of fluorescent signal et al, 1995; et al, 1995). to the sensitivity is amplification of the This to the would fluorescent detection of mutations in nuclei. amplification has been with some using extended DNA from but at this stage not or on et al, 1998). In the relatively time its FISH has had a major impact on cytogenetic analysis, due to the sensitivity and of its Although some of the applications will research the and probes for most cytogenetic abnormalities are now the of most clinical laboratories. However, conventional FISH can only provide to the specific and some of the The recent of FISH to the visualization of the entire human genome in different colours has the of and The of this approach is the ability to the whole genome in a single hybridization the screening of cytogenetics with the accuracy of molecular The belief that cytogenetics is more an than a has been from the the aid of new colour techniques and cytogenetic analysis now a molecular of The major impact of this development in field of haematological is to be the identification of new and non-random chromosome rearrangements and clinical of the most recent innovations to The most of fluorescent metaphase is now by and and will not only the of such but the sensitivity of interphase FISH analysis. many of the of cytogenetic analysis by the future for cytogenetics has The all of the of the particularly for the and analysis of the cell of the described here was by the and the