Targeting Antiapoptotic Genes Upregulated by Androgen Withdrawal Using Antisense Oligodeoxynucleotides to Enhance Androgen-and Chemo-Sensitivity in Prostate Cancer

Androgen withdrawal is the only effective form of systemic therapy for men with advanced disease, producing symptomatic and/or objective response in 80% of patients. Unfortunately, androgen independent (AI) progression and death occurs within a few years in the majority of these cases (6). Prostate cancer is highly chemoresistant, with objective response rates of 10% and no demonstrated survival benefit (28). Hormone refractory prostate cancer (HRPC) is therefore the main obstacle to improving the survival and quality of life in patients with advanced disease, and novel therapeutic strategies that target the molecular basis of androgen and chemoresistance are required.

A high-density consensus map of barley linking DArT markers to SSR, RFLP and STS loci and agricultural traits

Background: Molecular marker technologies are undergoing a transition from largely serial assays measuring DNA fragment sizes to hybridization-based technologies with high multiplexing levels. Diversity Arrays Technology (DArT) is a hybridization-based technology that is increasingly being adopted by barley researchers. There is a need to integrate the information generated by DArT with previous data produced with gel-based marker technologies. The goal of this study was to build a high-density consensus linkage map from the combined datasets of ten populations, most of which were simultaneously typed with DArT and Simple Sequence Repeat (SSR), Restriction Enzyme Fragment Polymorphism (RFLP) and/or Sequence Tagged Site (STS) markers. Results: The consensus map, built using a combination of JoinMap 3.0 software and several purpose-built perl scripts, comprised 2,935 loci (2,085 DArT, 850 other loci) and spanned 1,161 cM. It contained a total of 1,629 'bins' (unique loci), with an average inter-bin distance of 0.7 ± 1.0 cM (median = 0.3 cM). More than 98% of the map could be covered with a single DArT assay. The arrangement of loci was very similar to, and almost as optimal as, the arrangement of loci in component maps built for individual populations. The locus order of a synthetic map derived from merging the component maps without considering the segregation data was only slightly inferior. The distribution of loci along chromosomes indicated centromeric suppression of recombination in all chromosomes except 5H. DArT markers appeared to have a moderate tendency toward hypomethylated, gene-rich regions in distal chromosome areas. On the average, 14 ± 9 DArT loci were identified within 5 cM on either side of SSR, RFLP or STS loci previously identified as linked to agricultural traits. Conclusion: Our barley consensus map provides a framework for transferring genetic information between different marker systems and for deploying DArT markers in molecular breeding schemes. The study also highlights the need for improved software for building consensus maps from high-density segregation data of multiple populations.

Decoration of Pasteurella multocida lipopolysaccharide with phosphocholine is important for virulence

Phosphocholine (PCho) is an important substituent of surface structures expressed by a number of bacterial pathogens. Its role in virulence has been investigated in several species, in which it has been shown to play a role in bacterial adhesion to mucosal surfaces, in resistance to antimicrobial peptides, or in sensitivity to complement-mediated killing. The lipopolysaccharide (LPS) structure of Pasteurella multocida strain Pm70, whose genome sequence is known, has recently been determined and does not contain PCho. However, LPS structures from the closely related, virulent P. multocida strains VP161 and X-73 were shown to contain PCho on their terminal galactose sugar residues. To determine if PCho was involved in the virulence of P. multocida, we used subtractive hybridization of the VP161 genome against the Pm70 genome to identify a four-gene locus (designated pcgDABC) which we show is required for the addition of the PCho residues to LPS. The proteins predicted to be encoded by pcgABC showed identity to proteins involved in choline uptake, phosphorylation, and nucleotide sugar activation of PCho. We constructed a P. multocida VP161 pcgC mutant and demonstrated that this strain produces LPS that lacks PCho on the terminal galactose residues. This pcgC mutant displayed reduced in vivo growth in a chicken infection model and was more sensitive to the chicken antimicrobial peptide fowlicidin-1 than the wild-type P. multocida strain

NEESTIMATOR v2: re-implementation of software for the estimation of contemporary effective population size (N-e) from genetic data

NeEstimator v2 is a completely revised and updated implementation of software that produces estimates of contemporary effective population size, using several different methods and a single input file. NeEstimator v2 includes three single-sample estimators (updated versions of the linkage disequilibrium and heterozygote-excess methods, and a new method based on molecular coancestry), as well as the two-sample (moment-based temporal) method. New features include the following: (i) an improved method for accounting for missing data; (ii) options for screening out rare alleles; (iii) confidence intervals for all methods; (iv) the ability to analyse data sets with large numbers of genetic markers (10000 or more); (v) options for batch processing large numbers of different data sets, which will facilitate cross-method comparisons using simulated data; and (vi) correction for temporal estimates when individuals sampled are not removed from the population (Plan I sampling). The user is given considerable control over input data and composition, and format of output files. The freely available software has a new JAVA interface and runs under MacOS, Linux and Windows.

THE CHROMOSOMES OF TUFTED DEER (ELAPHODUS-CEPHALOPHUS)

Mitotic and meiotic chromosome preparations of the tufted deer (Elaphodus cephalophus) were studied to elucidate the sex-chromosomal polymorphism evidenced by this species. Females had 2n = 46 or 47 chromosomes, whereas males had 2n = 47 or 48 chromosomes. An X;autosome translocation was identified by synaptonemal complex analysis of spermatocytes at pachytene and confirmed by the presence of a trivalent at diakinesis/metaphase I. The present work, in combination with earlier observations by others, indicates that E. cephalophus possesses a varied X-chromosome morphology involving an X;autosome translocation and addition of varying amounts of heterochromatin. It is speculated that sex-chromosome polymorphism may be responsible for the observed differences in diploid chromosome number of tufted deer.

A COMPARATIVE-STUDY OF KARYOTYPES OF MUNTJACS BY CHROMOSOME PAINTING

We have used a combination of chromosome sorting, degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), chromosome painting and digital image capturing and processing techniques for comparative chromosome analysis of members of the genus Muntiacus. Chromosome-specific ''paints'' from a female Indian muntjac were hybridised to the metaphase chromosomes of the Gongshan, Black, and Chinese muntjac by both single and three colour chromosome painting. Karyotypes and idiograms for the Indian, Gongshan, Black and Chinese muntjac were constructed, based on enhanced 4', 6-diamidino-2-phenylindole (DAPI) banding patterns. The hybridisation signal for each paint was assigned to specific bands or chromosomes for all of the above muntjac species. The interspecific chromosomal homology was demonstrated by the use of both enhanced DAPI banding and comparative chromosome painting. These results provide direct molecular cytogenetic evidence for the tandem fusion theory of the chromosome evolution of muntjac species.

Evolution of the black muntjac (Muntiacus crinifrons) karyotype revealed by comparative chromosome painting

The black muntjac (Muntiacus crinifrons) has an unusual karyotype of 2n = 8 in females and 2n = 9 in males. We have studied the evolution of this karyotype by hybridising chromosome-specific paints derived from flow-sorted chromosomes of the Chinese muntjac (M. reevesi, 2n = 46) to chromosomes of the black muntjac. The hybridisation pattern allowed us to infer chromosomal homologies between these two species. Tandem and centromeric fusions, reciprocal translocations, and insertions are involved in the reduction of the diploid number from 2n = 46 to 2n = 8, 9. The painting patterns further show complex chromosomal rearrangements in the male black muntjac which involve more than half the karyotype, including both sex chromosomes. Since early meiosis is reported to be normal without any visible inversion loops of the synaptonemal complex, the observed chromosomal rearrangements would lead to heterosynapsis and, therefore, leave a large fraction of the male black muntjac karyotype balanced between the two sexes.

Mapping chromosomal homologies between humans and two langurs (Semnopithecus francoisi and S-Phayrei) by chromosome painting

Chromosomal homologies were established between human and two Chinese langurs (Semnopithecus francoisi, 2n=44, and S. phayrei, 2n=44) by chromosome painting with chromosome-specific DNA probes of all human chromosomes except the Y. Both langur species showed identical hybridization patterns in addition to similar G-banding patterns. In total, 23 human chromosome-specific probes detected 30 homologous chromosome segments in a haploid langur genome. Except for human chromosomes 1, 2, 6, 16 and 19 probes, which each gave signals on two non-homologous langur chromosomes respectively, all other probes each hybridized to a single chromosome. The results indicate a high degree of conservation of chromosomal synteny between human and these two Chinese langurs. The human chromosome 2 probe painted the entire euchromatic regions of langur chromosomes 14 and 19. Human chromosome 1 probe hybridized to three regions on langur autosomes, one region on langur chromosome 4 and two regions on langur chromosome 5. Human 19 probe hybridized on the same pattern to one region on chromosome 4 and to two regions on langur chromosome 5, where it alternated with the human chromosome 1 probe. Human 6 and 16 probes both hybridized to one region on each of the two langur autosomes 15 and 18. Only two langur chromosomes (12 and 21) were each labelled by probes specific for two whole human chromosomes (14 and 15 and 21 and 22 respectively). Comparison of the hybridization patterns of human painting probes on these two langurs with the data on other Old World primates suggests that reciprocal and Robertsonian translocations as will as inversions could have occurred since the divergance of human and the langurs from a common ancestor. This comparison also indicates that Asian colobines are karyotypically more closely related to each other that to African colobines.

Reciprocal chromosome painting illuminates the history of genome evolution of the domestic cat, dog and human

Domestic cats and dogs are important companion animals and model animals in biomedical research. The cat has a highly conserved karyotype, closely resembling the ancestral karyotype of mammals, while the dog has one of the most extensively rearranged mammalian karyotypes investigated so far. We have constructed the first detailed comparative chromosome map of the domestic dog and cat by reciprocal chromosome painting. Dog paints specific for the 38 autosomes and the X chromosomes delineated 68 conserved chromosomal segments in the cat, while reverse painting of cat probes onto red fox and dog chromosomes revealed 65 conserved segments. Most conserved segments on cat chromosomes also show a high degree of conservation in G-banding patterns compared with their canine counterparts. At least 47 chromosomal fissions (breaks), 25 fusions and one inversion are needed to convert the cat karyotype to that of the dog, confirming that extensive chromosome rearrangements differentiate the karyotypes of the cat and dog. Comparative analysis of the distribution patterns of conserved segments defined by dog paints on cat and human chromosomes has refined the human/cat comparative genome map and, most importantly, has revealed 15 cryptic inversions in seven large chromosomal regions of conserved synteny between humans and cats.

Dog chromosome-specific paints reveal evolutionary inter- and intrachromosomal rearrangements in the American mink and human

Forty chromosome-specific paint probes of the domestic dog (Canis familiaris, 2n = 78) were used to delineate conserved segments on metaphase chromosomes of the American mink (Mustela vison, 2n = 30) by fluorescence in situ hybridisation. Half of the 38 canine autosomal probes each painted one pair of homologous segments in a diploid mink metaphase, whereas the other 19 dog probes each painted from two to five pairs of discrete segments. In total, 38 canine autosomal paints highlighted 71 pairs of conserved segments in the mink. These painting results allow us to establish a complete comparative chromosome map between the American mink and domestic dog. This map demonstrates that extensive chromosome rearrangements differentiate the karyotypes of the dog and American mink. The 38 dog autosomes could be reconstructed from the 14 autosomes of the American mink through at least 47 fissions, 25 chromosome fusions, and six inversions. Furthermore, comparison of the current dog/mink map with the published human/dog map discloses 23 cryptic intrachromosomal rearrangements in 10 regions of conserved synteny in the human and American mink genomes and thus further refined the human/mink comparative genome map. Copyright (C) 2000 S. Karger AG, Basel.

Conserved chromosome segments in Hylobates hoolock revealed by human and H-leucogenys paint probes

A complete comparative chromosome map of the white-browed gibbon (Hylobates hoolock, 2n = 38), white-cheeked gibbon (Hylobates leucogenys, 2n = 52), and human has been established by hybridising H. leucogenys chromosome-specific paints and human 24-colour paints onto H. hoolock metaphase chromosomes. In the 18 H. hoolock autosomes, we identified 62 conserved segments that showed DNA homology to regions of the 25 H. leucogenys autosomes, Numerous interchromosomal rearrangements differentiate the karyotypes of H. leucogenys and H. hoolock. Only H. hoolock chromosome 10 showed homology to one entire autosome of H. leucogenys. The hybridisation of human 24-colour paints not only confirmed most of the chromosome correspondences between human and H. hoolock established previously but also helped to correct five erroneous assignments and revealed three new segments. Our results demonstrate that the karyotypes of the extant gibbons have arisen mainly through extensive translocation events and that the karyotype of H. hoolock more closely resembles the ancestral karyotype of Hylobates, rather than the karyotype of H. leucogenys. Copyright (C) 2001 S. Karger AG, Basel.

Phylogenetic implications of the 38 putative ancestral chromosome segments for four canid species

Chromosome homologies between the Japanese raccoon dog (Nectereutes procyonoides viverrinus, 2n = 39 + 2-4 B chromosomes) and domestic dog (Canis familiaris, 2n = 78) have been established by hybridizing a complete set of canine paint probes onto high-res

Comparative molecular cytogenetic studies in the order Carnivora: mapping chromosomal rearrangements onto the phylogenetic tree

We have made a set of chromosome-specific painting probes for the American mink by degenerate oligonucleotide primed-PCR (DOP-PCR) amplification of flow-sorted chromosomes. The painting probes were used to delimit homologous chromosomal segments among human, red fox, dog, cat and eight species of the family Mustelidae, including the European mink, steppe and forest polecats, least weasel, mountain weasel, Japanese sable, striped polecat, and badger. Based on the results of chromosome painting and G-banding, comparative maps between these species have been established. The integrated map demonstrates a high level of karyotype conservation among mustelid species. Comparative analysis of the conserved chromosomal segments among mustelids and outgroup species revealed 18 putative ancestral autosomal segments that probably represent the ancestral chromosomes, or chromosome arms, in the karyotype of the most recent ancestor of the family Mustelidae. The proposed 2n = 38 ancestral Mustelidae karyotype appears to have been retained in some modern mustelids, e.g., Martes, Lutra, ktonyx, and Vormela. The derivation of the mustelid karyotypes from the putative ancestral state resulted from centric fusions, fissions, the addition of heterochromatic arms, and occasional pericentric inversions. Our results confirm many of the evolutionary conclusions suggested by other data and strengthen the topology of the carnivore phylogenetic tree through the inclusion of genome-wide chromosome rearrangements. Copyright (C) 2002 S. KargerAG, Basel.

Karyotypic relationships of horses and zebras: results of cross-species chromosome painting

Complete sets of chromosome-specific painting probes, derived from flow-sorted chromosomes of human (HSA), Equus caballus (ECA) and Equus burchelli (EBU) were used to delineate conserved chromosomal segments between human and Equits burchelli, and among four equid species, E. przewalskii (EPR), E. caballus, E. burchelli and E. zebra hartmannae (EZH) by cross-species chromosome painting. Genome-wide comparative maps between these species have been established. Twenty-two human autosomal probes revealed 48 conserved segments in E. burchelli. The adjacent segment combinations HSA3/21, 7/16p, 16q/19q, 14/15, 12/22 and 4/8, presumed ancestral syntenies for all eutherian mammals, were also found conserved in E. burchelli. The comparative maps of equids allow for the unequivocal characterization of chromosomal rearrangements that differentiate the karyotypes of these equid species. The karyotypes of E. przewalskii and E. caballus differ by one Robertsonian translocation (ECA5 = EPR23 + EPR24); numerous Robertsonian translocations and tandem fusions and several inversions account for the karyotypic differences between the horses and zebras. Our results shed new light on the karyotypic evolution of Equidae. Copyright (C) 2003 S. Karger AG, Basel.

Cross-species chromosome painting in the Perissodactyla: delimitation of homologous regions in Burchell's zebra (Equus burchellii) and the white (Ceratotherium simum) and black rhinoceros (Diceros bicornis)

Conserved chromosomal segments in the black rhinoceros, Diceros bicornis (DB1, 2n = 84), and its African sister-species the white rhinoceros, Ceratotherim simum (CSI, 2n = 82), were detected using Burchell's zebra (Equus burchellii, EBU, 2n = 44) chromosome-specific painting probes supplemented by a subset of those developed for the horse (Equus caballus, ECA, 2n = 64). In total 41 and 42 conserved autosomal segments were identified in C simum and D. bicornis respectively. Only 21 rearrangements (20 fissions and I fusion) are necessary to convert the Burchell's zebra karyotype into that of the white rhinoceros. One fission distinguishes the D. bicornis and C simum karyotypes which, excluding hetero- chromatic differences, are identical in all respects at this level of resolution. Most Burchell's zebra chromosomes correspond to two rhinoceros chromosomes although in four instances (EBU 18, 19, 20 and 21) whole chromosome synteny has been retained among these species. In contrast, one rhinoceros chromosome (DBI1, CSI1) comprises two separate Burchell's zebra chromosomes (EBU11 and EBU17). In spite of the high diploid numbers of the two rhinoceros species their karyotypes are surprisingly conserved offering a glimpse of the putative ancestral perissodactyl condition and a broader understanding of genome organization in mammals. Copyright (C) 2003 S. Karger AG, Base