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Familial hypertrophic cardiomyopathy (HCM or CMH) is a myocardial disorder caused by mutations that affect the contractile machinery of heart muscle cells. Genetic testing of HCM patients is hampered by the fact that mutations in at least eight different genes contribute to the disease. An affordable high‐throughput mutation detection method is as yet not available. Since a significant number of mutations have been repeatedly found in unrelated families, we consider it feasible to pre‐screen patients for known mutations, before more laborious techniques capable of detecting new mutations are applied. Here we demonstrate that the principle of hybridization of DNA to oligonucleotide probes immobilized on chips (glass slides) can be applied for this purpose. We have developed a low‐density oligonucleotide probe array capable of detecting 12 different heterozygous mutations (in four different genes), among them single‐ and double‐base exchanges, a single nucleotide insertion, and a trinucleotide deletion. The assay is simple and may be amenable to automation. Detection is achieved with a CCD camera‐based fluorescence biochip reader. The technique turned out to be robust: Variations in either the relative position of a mutation, or the amount and size of target‐DNA were compatible with mutation detection. Mutations could even be detected in amplicons as long as 800 bp, allowing the screening of more than one exon in one amplicon. Our data suggest that the development of a chip that covers all or most of known HCM‐associated mutations is feasible and useful. Hum Mutat 19:560–569, 2002. © 2002 Wiley‐Liss, Inc.

The X and Y Chromosomes (Chrs) of eutherian (“placental”) mammals share a pseudo-autosomal region (PAR) that pairs and recombines at meiosis. In humans and other eutherians, the PAR contains several active genes and has also been thought to be critical for pairing and fertility. In order to explore the origin of the PAR, we cloned and mapped three human or mouse pseudoautosomal genes in marsupials, a group of mammals that diverged from eutherians about 130 (MYrBP). All three genes were autosomal in marsupials, and two co-localized with other human Xp genes on an autosome. This implies that the human PAR, like most of human Xp, represents a relic of an autosomal region added to both X and Y Chrs between 80 and 150 MYrBP.

Cross-species chromosome painting was used to investigate genome rearrangements between tammar wallaby Macropus eugenii (2n = 16) and the swamp wallaby Wallabia bicolor (2n = 10♀/11♂), which diverged about 6 million years ago. The swamp wallaby has an XX female:XY₁Y₂ male sex chromosome system thought to have resulted from a fusion between an autosome and the small original X, not involving the Y. Thus, the small Y₁ should represent the original Y and the large Y₂ the original autosome. DNA paints were prepared from flow-sorted and micro-dissected chromosomes from the tammar wallaby. Painting swamp wallaby spreads with each tammar chromosome-specific probe gave extremely strong and clear signals in single-, two-, and three-color FISH. These showed that two tammar wallaby autosomes are represented unchanged in the swamp wallaby, two are represented by different centric fusions, and one by a tandem fusion to make the very long arms of swamp wallaby Chromosome (Chr) 1. The large swamp wallaby X comprises the tammar X as its short arm, and a tandemly fused 7 and 2 as the long arm. The acrocentric swamp wallaby Y₂ is a 2/7 fusion, homologous with the long arm of the X. The small swamp wallaby Y₁ is confirmed as the original Y by its painting with the tammar Y. However, the presence of sequences shared between the microdissected tammar Xp and Y on the swamp wallaby Y₂ implies that the formation of the compound sex chromosomes involved addition of autosome(s) to both the original X and Y. We propose that this involved fusion with an ancient pseudoautosomal region followed by fission proximal to this shared region.

Marsupial sex chromosomes are smaller than their eutherian counterparts and are thought to reflect an ancestral mammalian X and Y. The gene content of this original X is represented largely by the long arm of the human X chromosome. Genes on the short arm of the human X are autosomal in marsupials and monotremes, and represent a recent addition to the eutherian X and Y. The marsupial X and Y apparently lack a pseudoautosomal region and show only end-to-end pairing at meiosis. However, the sex chromosomes of macropodid marsupials (kangaroos and wallabies) are larger than the sex chromosomes of other groups, and a nucleolus organizer is present on the X and occasionally the Y. Chromosome painting using DNA from sorted and microdissected wallaby X and Y chromosomes reveals homologous sequences on the tammar X and Y chromosomes, concentrated on the long arm of the Y chromosome and short arm of the X. Ribosomal DNA sequences were detected by fluorescence in situ hybridization on the wallaby Xp but not the Y. Since no chiasmata have been observed in marsupial sex chromosomes, it is unlikely that these shared sequences act as a pseudoautosomal region within which crossing over may occur, but they may be required for end-to-end associations. The shared region of wallaby X and Y chromosomes bears no homology with the recently added region of the eutherian sex chromosomes, so we conclude that independent additions occurred to both sex chromosomes in a eutherian and macropodid ancestor, as predicted by the addition-attrition hypothesis of sex chromosome evolution.

The sequencing of the entire human genome was completed in June 2000. The sequence, however, is only a starting point and gene-function is now of major interest. All this information shows that gene-based diagnostics can be helpful for treatment targeting and patient surveillance. High volume gene expression assays can optimize pharmaceutical therapies by targeting genome-based treatments to specific patient populations and providing methods to study genes involved with cancer growth patterns and tumor suppression. Molecular biology, so far, has elucidated many of the genetic mechanisms underlying heritable metabolic diseases, so that appropriate diagnostic assays will revolutionize molecular diagnostics in medicine and pharmaceuticals. One of the most promising new technologies designed to analyze large amounts of genomic information rapidly is the DNA chip.

Interspecies chromosome painting has been used to demonstrate homologies between chromosomes of human and other primates (Wienberg et al. 1990), carnivores (Rettenberger et al. 1995), and artiodactyls (Solinas-Toldo et al. 1995), which diverged about 60 million years before present (MyrBP). However, there has been no success in using the technique for wider comparisons, for instance between eutherian (\"placental\") mammals and marsupials, which diverged about 130 MyrBP. We have now used a paint derived from the X Chromosome (Chr) flow-sorted from the tammar wallaby (Macropus eugenii), to detect homologous regions on chromosomes from cultured lymphocytes from a human male. Chromosome painting was performed under our usual conditions (Toder et al. 1997) with a 3-day hybridization time. The X Chr paint, labeled with super(32)P, was used to probe a Southern blot containing wallaby and human DNA to show that there was no DNA contaminant that could have influenced the hybridization results. Chromosome painting revealed strong signal on the long arm of the X Chr and proximally on the short arm (Fig. 1). No signal was detected on Xp distal to about Xp11.2, or on any autosomes. Paints derived from wallaby autosomes and the Y Chr detected no signal on human chromosome spreads under the same conditions.

The mammalian X and Y chromosomes are very different in size and gene content. The Y chromosome is much smaller than the X and consists largely of highly repeated non-coding DNA, containing few active genes. The 65-Mb human Y is homologous to the X over two small pseudoautosomal regions which together contain 13 active genes. The heterochromatic distal half of the human Yq is entirely composed of highly repeated non-coding DNA, and even the euchromatic portion of the differential region is largely composed of non-coding repeated sequences, amongst which about 30 active genes are located. The basic marsupial Y chromosome (about 10 Mb) is much smaller than that of humans or other eutherian mammals. It appears to include no PAR, since it does not undergo homologous pairing, synaptonemal complex formation or recombination with the X. We show here that the tiny dunnart Y chromosome does not share cytogenetically detectable sequences with any other chromosome, suggesting that it contains many fewer repetitive DNA sequences than the human or mouse Y chromosomes. However, it shares several genes with the human and/or mouse Y chromosome, including the sex determining gene SRY and the candidate spermatogenesis gene RBMY, implying that the marsupial and eutherian Y are monophyletic. This minimal mammalian Y chromosome might provide a good model Y in which to hunt for new mammalian Y specific genes.

The three human male specific expressed gene families DAZ, RBM, and TSPY are known to be repetitively clustered in the Y-specific region of the human Y Chromosome (Chr). RBM and TSPY are Y-specifically conserved in simians, whereas DAZ cannot be detected on the Y chromosomes of New World monkeys. The proximity of SRY to the pseudoautosomal region (PAR) is highly conserved and thus most effectively stabilizes the pseudoautosomal boundary on the Y (PABY) in simians. In contrast, the non-recombining part of the Y Chrs, including DAZ, RBM, and TSPY, was exposed to species-specific amplifications, diversifications, and rearrangements. Evolutionary fast fixation of any of these variations was possible as long as they did not interfere with male fertility.

The genomic nucleotide sequence and chromosomal position of the interleukin 5 (IL5) gene has been described for the model marsupial Macropus eugenii (tammar wallaby). A 272 base pair genomic IL5 polymerase chain reaction (PCR) product spanning exon 3, intron 3, and exon 4 was generated using stripe-faced dunnart (Sminthopsis macroura) DNA. This PCR product was used to isolate a genomic lambda clone containing the complete IL5 gene from a tammar wallaby EMBL3 lambda library. Sequencing revealed that the tammar wallaby IL5 gene consists of four exons separated by three introns. Comparison of the marsupial coding sequence with coding sequences from eutherian species revealed 61 to 69% identity at the nucleotide level and 48 to 63% identity at the amino acid (aa) level. A polymorphic complex compound microsatellite was identified within intron 2 of the tammar wallaby IL5 gene. This microsatellite was also found in other marsupials including the swamp wallaby, tree kangaroo, stripe-faced dunnart, South American opossum, brushtail possum, and koala. Fluorescence in situ hybridization using DNA from the IL5 clone on tammar wallaby chromosomes indicated that the IL5 gene is located on Chromosome 1.