Comparative molecular cytogenetics in cetartiodactyla

Cetartiodactyla comprises Artiodactyla (even-toed ungulates) and Cetacea (whales, dolphins and porpoises). Artiodactyla is a large taxon represented by about 200 living species ranked in 10 families. Cetacea are classified into 13 families with almost 80 species. Many publications concerning karyotypic relationships in Cetartiodactyla have been published in previous decades. Formerly, the karyotypes of closely related species were compared by chromosome banding. Introduction of molecular cytogenetic methods facilitated comparative mapping between species with highly rearranged karyotypes and distantly related species. Such information is a prerequisite for the understanding of karyotypic phylogeny and the reconstruction of the karyotypes of common ancestors. This study summarizes the data on chromosome evolution in Cetartiodactyla, mainly derived from molecular cytogenetic studies. Traditionally, phylogenetic relationships of most groups have been estimated using morphological data. However, the results of some molecular studies of mammalian phylogeny are discordant with traditional conceptions of phylogeny. Cetartiodactyls provide several examples of incongruence between traditional morphological and molecular data. Such cases of conflict include the relationships of the major clades of artiodactyls, the relationships among the extant families of the suborder Ruminantia or the phylogeny of the family Bovidae. The most unexpected aspect of the molecular phylogeny was the recognition that Cetacea is a deeply nested member of Artiodactyla. The largest living order of terrestrial hoofed mammals is the even-toed hoofed mammals, or Artiodactyla. The artiodactyls are composed of over 190 living species including pigs, peccaries, hippos, camels, llamas, deer, pronghorns, giraffes, sheep, goats, cattle and antelopes. Cetacea is an order of wholly aquatic mammals, which include whales, dolphins and porpoises. Cetartiodactyla has become the generally accepted name for the clade containing both of these orders.     

Applications of fluorescence spectroscopy to molecular cytogenetics

Phenomena studied by fluorescence spectroscopy, including differential perturbations of dye fluorescence by variations in DNA composition and interdye energy transfer, can be applied to cytological studies of chromosome structure, replication, and repair. When combined with fluorescence-activated flow sorting, fluorescent differentiation between metaphase chromosomes can be exploited to obtain recombinant DNA libraries enriched for all or parts of individual chromosomes. Cloned DNA segments from such libraries continue to find application in studies of human chromosome structure and function and in molecular linkage analyses, thus leading to molecular genetic investigations of human disease states.     

Diagnosis of minor chromosome modifications by molecular cytogenetics

Chromosomal in situ hybridization with radioactive probes allows the detection of single copy DNA segments of very small size. In two phenotypically normal individuals, classical chromosomal analysis revealed a small partial deletion of chromosome 18 short arm. In situ hybridization of probe D18S3, located at 18p13, showed in both instances a balanced reciprocal translocation: 46,XY,t(5;18) (p153;p112) and 46,XX,t(18;22)(p111;p11), respectively.     

Elongated mouse chromosomes suitable for enhanced molecular cytogenetics

Characterization of genetic disorders in humans and animal models requires identification of chromosomal aberrations. However, identifying fine deletions or insertion in metaphase chromosomes has been always a challenge due to limitations of resolution. In this study we developed a rapid method for chromosome elongation using two different intercalating agents: ethidium bromide and 5-bromo-2′-deoxyuridine (BrdU), together with a short-term mitotic block using colcemid. About 70% of the chromosomes from cells that underwent this elongation procedure reached three times longer than those prepared from control cells. FISH experiments using elongated chromosomes revealed a duplicated region of chromosome 11 that was not visible in cells prepared with conventional methods.     

Characterization of the Nicotiana tabacum L. genome by molecular cytogenetics

Nicotiana tabacum (2n=48) is a natural amphidiploid with component genomes S and T. We used non-radioactive in situ hybridization to provide physical chromosome markers for N. tabacum, and to determine the extant species most similar to the S and T genomes. Chromosomes of the S genome hybridized strongly to biotinylated total DNA from N. sylvestris, and showed the same physical localization of a tandemly repeated DNA sequence, HRS 60.1, confirming the close relationship between the S genome and N. sylvesfris. Results of dot blot and in situ hybridizations of N. tabacum DNA to biotinylated total genomic DNA from N. tomentosiformis and N. otophora suggested that the T genome may derive from an introgressive hybrid between these two species. Moreover, a comparison of nucleolus-organizing chromosomes revealed that the nucleolus organizer region (NOR) most strongly expressed in N. tabacum had a very similar counterpart in N. otophora. Three different N. tabacum genotypes each had up to 9 homozygous translocations between chromosomes of the S and T genomes. Such translocations, which were either unilateral or reciprocal, demonstrate that intergenomic transfer of DNA has occurred in the amphidiploid, possibly accounting for some results of previous genetic and molecular analyses. Molecular cytogenetics of N. tabacum has identified new chromosome markers, providing a basis for physical gene mapping and showing that the amphidiploid genome has diverged structurally from its ancestral components.     

Characterization and application of soybean YACs to molecular cytogenetics

Yeast artificial chromosomes (YACs) are widely used in the physical analysis of complex genomes. In addition to their value in chromosome walking for map-based cloning, YACs represent excellent probes for chromosome mapping using fluorescence in situ hybridization (FISH). We have screened such a library for low-copy-number clones by hybridization to total genomic DNA. Four clones were chosen for chromosome tagging based upon their low or moderate signal. By using degenerate oligonucleotide-primed PCR (DOP-PCR), we were able to use relatively small amounts of soybean YAC DNA, isolated directly by preparative pulsed-field gel electrophoresis, as FISH probes for both metaphase chromosome spreads and interphase nuclei. FISH chromosomal analysis using the three of the clones as probes resulted in relatively simple hybridization patterns consistent with a single homologous locus or two homoeologous loci. The fourth YAC probe resulted in a diffuse hybridization pattern with signal on all metaphase chromosomes. We conclude that YACs represent a valuable source of probes for chromosomal analysis in soybean.     

Characterising chromosome rearrangements: recent technical advances in molecular cytogenetics

Genomic rearrangements can result in losses, amplifications, translocations and inversions of DNA fragments thereby modifying genome architecture, and potentially having clinical consequences. Many genomic disorders caused by structural variation have initially been uncovered by early cytogenetic methods. The last decade has seen significant progression in molecular cytogenetic techniques, allowing rapid and precise detection of structural rearrangements on a whole-genome scale. The high resolution attainable with these recently developed techniques has also uncovered the role of structural variants in normal genetic variation alongside single-nucleotide polymorphisms (SNPs). We describe how array-based comparative genomic hybridisation, SNP arrays, array painting and next-generation sequencing analytical methods (read depth, read pair and split read) allow the extensive characterisation of chromosome rearrangements in human genomes.     

The evolution of the avian genome as revealed by comparative molecular cytogenetics

Birds are characterised by feathers, flight, a small genome and a very distinctive karyotype. Despite the large numbers of chromosomes, the diploid count of 2n approximately 80 has remained remarkably constant with 63% of birds where 2n = 74-86, 24% with 2n = 66-74 and extremes of 2n = 40 and 2n = 142. Of these, the most studied is the chicken (2n = 78), and molecular cytogenetic probes generated from this species have been used to further understand the evolution of the avian genome. The ancestral karyotype is, it appears, very similar to that of the chicken, with chicken chromosomes 1, 2, 3, 4q, 5, 6, 7, 8, 9, 4p and Z representing the ancestral avian chromosomes 1-10 + Z; chromosome 4 being the most ancient. Avian evolution occurred primarily in three stages: the divergence of the group represented by extant ratites (emu, ostrich etc.) from the rest; divergence of the Galloanserae (chicken, turkey, duck, goose etc.)--the most studied group; and divergence of the 'land' and 'water' higher birds. Other than sex chromosome differentiation in the first divergence there are no specific changes associated with any of these evolutionary milestones although certain families and orders have undergone multiple fusions (and some fissions), which has reduced their chromosome number; the Falconiformes are the best described. Most changes, overall, seem to involve chromosomes 1, 2, 4, 10 and Z where the Z changes are intrachromosomal; there are also some recurring (convergent) events. Of these, the most puzzling involves chromosomes 4 and 10, which appear to have undergone multiple fissions and/or fusions throughout evolution - three possible hypotheses are presented to explain the findings. We conclude by speculating as to the reasons for the strange behaviour of these chromosomes as well as the role of telomeres and nuclear organisation in avian evolution.     

Methods of molecular cytogenetics for studying interphase chromosomes in human brain cells

One of the main genetic factors determining the functional activity of the genome in somatic cells, including brain nerve cells, is the spatial organization of chromosomes in the interphase nucleus. For a long time, no studies of human brain cells were carried out until high-resolution methods of molecular cytogenetics were developed to analyze interphase chromosomes in nondividing somatic cells. The purpose of the present work was to assess the potential of high-resolution methods of interphase molecular cytogenetics for studying chromosomes and the nuclear organization in postmitotic brain cells. A high efficiency was shown by such methods as multiprobe and quantitative fluorescence in situ hybridization (Multiprobe FISH and QFISH), ImmunoMFISH (analysis of the chromosome organization in different types of brain cells), and interphase chromosome-specific multicolor banding (ICS-MCB). These approaches allowed studying the nuclear organization depending on the gene composition and types of repetitive DNA of specific chromosome regions in certain types of brain cells (in neurons and glial cells, in particular). The present work demonstrates a high potential of interphase molecular cytogenetics for studying the structural and functional organizations of the cell nucleus in highly differentiated nerve cells. Analysis of interphase chromosomes of brain cells in the normal and pathological states can be considered as a promising line of research in modern molecular cytogenetics and cell neurobiology, i. e., molecular neurocytogenetics.     

Application of molecular cytogenetics for chromosomal evolution of the Lemuriformes (Prosimians)

R-banding chromosomal studies of 21 species of Lemuriformes allowed us to reconstruct the presumed ancestral karyotype of all the Lemuriformes except for Daubentoniidae and permitted the construction of their phylogenetic tree. Chromosome painting with fluorescently labeled heterologous DNA probes permitted comparative chromosome maps to be established. The Zoo-FISH method was used to reassess the karyotypes of 22 species or subspecies. While our results largely confirm the previous reconstruction of the ancestral karyotype, they resulted in a modification of the previously established phylogenetic tree. The Daubentoniidae emerged first followed by the divergence of the families Cheirogaleidae, Indriidae, Lepilemuridae and Lemuridae. Eight chromosome rearrangements occurred in all Lemuriformes except for Daubentoniidae in the common trunk. The present findings do not allow us to propose the occurrence of any rearrangement common to Daubentoniidae and other Lemuriformes, and probably other Prosimii. Conserved syntenies previously described in various mammalian orders were also conserved, while others were specific to the Lemuriformes.     

Characterization of the Nicotiana tabacum L. genome by molecular cytogenetics

Nicotiana tabacum (2n=48) is a natural amphidiploid with component genomes S and T. We used non-radioactive in situ hybridization to provide physical chromosome markers for N. tabacum, and to determine the extant species most similar to the S and T genomes. Chromosomes of the S genome hybridized strongly to biotinylated total DNA from N. sylvestris, and showed the same physical localization of a tandemly repeated DNA sequence, HRS 60.1, confirming the close relationship between the S genome and N. sylvesfris. Results of dot blot and in situ hybridizations of N. tabacum DNA to biotinylated total genomic DNA from N. tomentosiformis and N. otophora suggested that the T genome may derive from an introgressive hybrid between these two species. Moreover, a comparison of nucleolus-organizing chromosomes revealed that the nucleolus organizer region (NOR) most strongly expressed in N. tabacum had a very similar counterpart in N. otophora. Three different N. tabacum genotypes each had up to 9 homozygous translocations between chromosomes of the S and T genomes. Such translocations, which were either unilateral or reciprocal, demonstrate that intergenomic transfer of DNA has occurred in the amphidiploid, possibly accounting for some results of previous genetic and molecular analyses. Molecular cytogenetics of N. tabacum has identified new chromosome markers, providing a basis for physical gene mapping and showing that the amphidiploid genome has diverged structurally from its ancestral components.     

Prader-Willi syndrome: clinical genetics, cytogenetics and molecular biology

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder that arises from lack of expression of paternally inherited genes known to be imprinted and located in the chromosome 15q11-q13 region. PWS is considered the most common syndromal cause of life-threatening obesity and is estimated at 1 in 10,000 to 20,000 individuals. A de novo paternally derived chromosome 15q11-q13 deletion is the cause of PWS in about 70% of cases, and maternal disomy 15 accounts for about 25% of cases. The remaining cases of PWS result either from genomic imprinting defects (microdeletions or epimutations) of the imprinting centre in the 15q11-q13 region or from chromosome 15 translocations. Here, we describe the clinical presentation of PWS, review the current understanding of causative cytogenetic and molecular genetic mechanisms, and discuss future directions for research.     

Relationships among 3 Kochia species based on PCR-generated molecular sequences and molecular cytogenetics.

Forage kochia (Kochia prostrata ssp. virescens 'Immigrant' is native to the arid and semiarid regions of central Eurasia. It was introduced into the United States in 1966 as PI 314929 and released as a perennial forage shrub in 1984. Kochia americana is a perennial native to the United States, whereas Kochia scorparia is an introduced annual species that became a weed. To assess both the breeding potential and the possibility of genetic contamination, relationships among the 3 Kochia species were analyzed using random amplified polymorphic DNA (RAPD) markers, sequence tagged site (STS) marker sequences of the chloroplast NADH dehydrogenase gene (ndhF), genomic in situ hybridization (GISH), and multicolor fluorescence in situ hybridization (MC-FISH). Seventy decamer random primers yielded 458 polymorphic bands from 9 plants of K. americana, 20 plants of K. prostrata, and 7 plants of K. scoparia. Fifty-four and 55 species-specific RAPD markers were identified for K. americana and K. prostrata, whereas 80 RAPD markers were specific to K. scoparia. Based on the presence or absence of informative RAPD markers, the 3 species always grouped into 3 distinct clusters in a NTSYSpc2.01b-generated dendrogram. The same relationships were found among the 3 Kochia species based on ndhF DNA sequence divergence. Using a set of 7 STS markers that can identify each Kochia species, we did not find a single interspecific hybrid from artificial hybridizations among the 3 Kochia species. In GISH studies, chromosomes of 1 species fluoresced in green only when they were probed by genomic DNA of the same species. Cross-hybridization by genomic DNA of another species was not observed. In FISH studies using pTa71 (for 18S-5.8S-26S rDNAs) and pScT7 (for 5S rDNA) as probes, there were 1, 1 and 3 pTa71 sites and 2, 1, and 1 pScT7 sites in each haplome of K. prostrata, K. americana, and K. scoparia, respectively. It is concluded that these 3 Kochia species are so genomically distinct that gene introgression among them would be extremely rare.