A strategy for the characterization of minute chromosome rearrangements using multiple color fluorescence in situ hybridization with chromosome-specific DNA libraries and YAC clones

The identification of marker chromosomes in clinical and tumor cytogenetics by chromosome banding analysis can create problems. In this study, we present a strategy to define minute chromosomal rearrangements by multicolor fluorescence in situ hybridization (FISH) with whole chromosome painting probes derived from chromosome-specific DNA libraries and Alu-polymerase chain reaction (PCR) products of various region-specific yeast artificial chromosome (YAC) clones. To demonstrate the usefulness of this strategy for the characterization of chromosome rearrangements unidentifiable by banding techniques, an 8p+ marker chromosome with two extra bands present in the karyotype of a child with multiple anomalies, malformations, and severe mental retardation was investigated. A series of seven-color FISH experiments with sets of fluorochrome-labeled DNA library probes from flow-sorted chromosomes demonstrated that the additional segment on 8p+ was derived from chromosome 6. For a more detailed characterization of the marker chromosome, three-color FISH experiments with library probes specific to chromosomes 6 and 8 were performed in combination with newly established telomeric and subtelomeric YAC clones from 6q25, 6p23, and 8p23. These experiments demonstrated a trisomy 6pter6p22 and a monosomy 8pter8p23 in the patient. The present limitations for a broad application of this strategy and its possible improvements are discusse

In silico analysis for the presence of HARDY an Arabidopsis drought tolerance DNA binding transcription factor product in chromosome 6 of Sorghum bicolor genome

Expression of the Arabidopsis HARDY (hrd) DNA binding transcription factor (555 bp present on chromosome 2) has been shown to increase WUE in rice by Karaba et al 2007 (PNAS, 104:15270–15275). We conducted a detail analysis of the complete sorghum genome for the similarity/presence of either DNA, mRNA or protein product of the Arabidopsis HARDY (hrd) DNA binding transcription factor (555 bp present on chromosome 2). Chromosome 6 showed a sequence match of 61.5 percent positive between 61 and 255 mRNA residues of the query region. Further confirmation was obtained by TBLASTN which showed that chromosome 6 of the sorghum genome has a region between 54948120 and 54948668 which has 80 amino acid similarities out of the 185 residues. A homology model was constructed and verified using Anolea, Gromos and Verify3D. Scanning the motif for possible activation sites revealed that there was a protein kinase C phosphorylation site between 15th and 20th residue. The study indicates the possibility of the presence of a DNA binding transcription factor in chromosome 6 of Sorghum bicolor with 60 percent similarity to that of Arabidopsis hrd DNA binding transcription factor

Smc5/6: a link between DNA repair and unidirectional replication?

Of the three structural maintenance of chromosome (SMC) complexes, two directly regulate chromosome dynamics. The third, Smc5/6, functions mainly in homologous recombination and in completing DNA replication. The literature suggests that Smc5/6 coordinates DNA repair, in part through post-translational modification of uncharacterized target proteins that can dictate their subcellular localization, and that Smc5/6 also functions to establish DNA-damage-dependent cohesion. A nucleolar-specific Smc5/6 function has been proposed because Smc5/6 yeast mutants display penetrant phenotypes of ribosomal DNA (rDNA) instability. rDNA repeats are replicated unidirectionally. Here, we propose that unidirectional replication, combined with global Smc5/6 functions, can explain the apparent rDNA specificity

Detection and Mapping of Quantitative Trait Loci that Determine Responsiveness

Exposure to 70% N2O evokes a robust antinociceptive effect in C57BL/6 (B6) but not in DBA/2 (D2) inbred mice. This study was conducted to identify quantitative trait loci (QTL) in the mouse genome that might determine responsiveness to N2O. Offspring from the F2 generation bred from B6 and D2 progenitors exhibited a broad range of responsiveness to N2O antinociception as determined by the acetic acid-induced abdominal constriction test. QTL analysis was then used to dissect this continuous trait distribution into component loci, and to map them to broad chromosomal regions. To this end, 24 spleens were collected from each of the following four groups: male and female F2 mice responding to 70% N2O in oxygen with 100% response (high-responders); and male and female F2 mice responding with 0% response (low-responders). Genomic DNA was extracted from the spleens and genotyped with simple sequence length polymorphism MapPairs markers. Findings were combined with findings from the earlier QTL analysis from BXD recombinant inbred mice [Brain Res 725 (1996) 23]. Combined results revealed two significant QTL that influence responsiveness to nitrous oxide on proximal chromosome 2 and distal chromosome 5, and one suggestive QTL on midchromosome 18. The chromosome 2 QTL was evident only in males. A significant interaction was found between a locus on chromosome 6 and another on chromosome 13 with a substantial effect on N2O antinociception

Hexavalents in spermatocytes of Robertsonian heterozygotes between Mus m. domesticus 2n 26 from the Vulcano and Lipari Islands (Aeolian Archipelago, Italy)

The size and shape of the chromosomes, as well as the chromosomal domains that compose them, are determinants in the distribution and interaction between the bivalents within the nucleus of spermatocytes in prophase I of meiosis. Thus the nuclear architecture characteristic of the karyotype of a species can be modified by chromosomal changes such as Robertsonian (RB) chromosomes. In this study we analysed the meiotic prophase nuclear organization of the heterozygous spermatocytes from Mus musculus domesticus 2n=26, and the synaptic configuration of the hexavalent formed by the dependent Rb chromosomes Rbs 6.16, 16.10, 10.15, 15.17 and the telocentric chromosomes 6 and 17. Spreads of 88 pachytene spermatocytes from two males were studied and in all of them five metacentric bivalents, four telocentric bivalents, one hexavalent and the XY bivalent were observed. About 48% of the hexavalents formed a chain or a ring of synapsed chromosomes, the latter closed by synapsis between the short arms of telocentric chromosomes 6 and 17. About 52% of hexavalents formed an open chain of 10 synapsed chromosomal arms belonging to 6 chromosomes. In about half of the unsynapsed hexavalents one of the telocentric chromosome short arms appears associated with the X chromosome single axis, which was otherwise normally paired with the Y chromosome. The cluster of pericentromeric heterochromatin mostly determines the hexavalent’s nuclear configuration, dragging the centromeric regions and all the chromosomes towards the nuclear envelope similar to an association of five telocentric bivalents. These reiterated encounters between these chromosomes restrict the interactions with other chromosomal domains and might favour eventual rearrangements within the metacentric, telocentric or hexavalent chromosome subsets. The unsynapsed short arms of telocentric chromosomes frequently bound to the single axis of the X chromosome could further complicate the already complex segregation of hexavalent chromosomes

Rapid generation of chromosome-specific alphoid DNA probes using the polymerase chain reaction

Non-isotopic in situ hybridization of chromosome-specific alphoid DNA probes has become a potent tool in the study of numerical aberrations of specific human chromosomes at all stages of the cell cycle. In this paper, we describe approaches for the rapid generation of such probes using the polymerase chain reaction (PCR), and demonstrate their chromosome specificity by fluorescence in situ hybridization to normal human metaphase spreads and interphase nuclei. Oligonucleotide primers for conserved regions of the alpha satellite monomer were used to generate chromosome-specific DNA probes from somatic hybrid cells containing various human chromosomes, and from DNA libraries from sorted human chromosomes. Oligonucleotide primers for chromosome-specific regions of the alpha satellite monomer were used to generate specific DNA probes for the pericentromeric heterochromatin of human chromosomes 1, 6, 7, 17 and X directly from human genomic DNA

Smc5/6 coordinates formation and resolution of joint molecules with chromosome morphology to ensure meiotic divisions

During meiosis, Structural Maintenance of Chromosome (SMC) complexes underpin two fundamental features of meiosis: homologous recombination and chromosome segregation. While meiotic functions of the cohesin and condensin complexes have been delineated, the role of the third SMC complex, Smc5/6, remains enigmatic. Here we identify specific, essential meiotic functions for the Smc5/6 complex in homologous recombination and the regulation of cohesin. We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms, respectively. Smc5/6 also localizes to recombination hotspots, where it promotes normal formation and resolution of a subset of joint-molecule intermediates. In this regard, Smc5/6 functions independently of the major crossover pathway defined by the MutLγ complex. Furthermore, we show that Smc5/6 is required for stable chromosomal localization of the XPF-family endonuclease, Mus81-Mms4Eme1. Our data suggest that the Smc5/6 complex is required for specific recombination and chromosomal processes throughout meiosis and that in its absence, attempts at cell division with unresolved joint molecules and residual cohesin lead to severe recombination-induced meiotic catastroph

Rad62 protein functionally and physically associates with the Smc5/Smc6 protein complex and is required for chromosome integrity and recombination repair in fission yeast

Smc5 and Smc6 proteins form a heterodimeric SMC (structural maintenance of chromosome) protein complex like SMC1-SMC3 cohesin and SMC2-SMC4 condensin, and they associate with non-SMC proteins Nse1 and Nse2 stably and Rad60 transiently. This multiprotein complex plays an essential role in maintaining chromosome integrity and repairing DNA double strand breaks (DSBs). This study characterizes a Schizosaccharomyces pombe mutant rad62-1, which is hypersensitive to methyl methanesulfonate (MMS) and synthetically lethal with rad2 (a feature of recombination mutants). rad62-1 is hypersensitive to UV and gamma rays, epistatic with rhp51, and defective in repair of DSBs. rad62 is essential for viability and genetically interacts with rad60, smc6, and brc1. Rad62 protein physically associates with the Smc5-6 complex. rad62-1 is synthetically lethal with mutations in the genes promoting recovery from stalled replication, such as rqh1, srs2, and mus81, and those involved in nucleotide excision repair like rad13 and rad16. These results suggest that Rad62, like Rad60, in conjunction with the Smc5-6 complex, plays an essential role in maintaining chromosome integrity and recovery from stalled replication by recombination