Fragile X chromosome and chromosome condensation

The effect of chromosome condensation on the frequency of expression of the fragile X chromosome was examined. Chromosome decondensation substances were tested for their ability to elicit expression or improve frequencies of expression of the fragile X chromosome in five patients. The substances tested included the AT specific DNA ligands ethidium bromide, Hoechst 33258, and netropsin, and the GC specific substances actinomycin D and olivomycin. Under culture conditions appropriate for eliciting fragile X expression none of the decondensation compounds studied significantly altered frequencies of expression, nor did any of the substances elicit fragile X expression under conditions that normally suppress fragile X expression. The fragile X was found to be more frequently evident in less condensed chromosome preparations from fibroblasts. The implications of these findings with respect to the nature of fragile sites are discussed.     

Mouse chromosome 19 and distal rat chromosome 1: a chromosome segment conserved in evolution

Through a combination of radiation hybrid mapping and studies by FISH and zoo-FISH we have made a comparative investigation of the distal portion of rat chromosome 1 (RNO1) and the entire mouse chromosome 19 (MMU19). It was found that homologous segments of RNO1 and MMU19 are similar in banding morphology and in length as determined by several different methods, and that the gene order of the 46 genes studied appears to be conserved across the homologous segments in the two species. High-resolution zoo-FISH techniques showed that MMU19 probes highlight only a continuous segment on RNO1 (1q43-qter), with no detectable signals on other rat chromosomes. We conclude that these data suggest the evolutionary conservation of a chromosomal segment from a common rodent ancestor. This segment now constitutes the entire MMU19 and a large segment distally on RNO1q in the mouse and rat, respectively.     

The three vibrio cholerae chromosome II-encoded ParE toxins degrade chromosome I following loss of chromosome II

Three homologues of the plasmid RK2 ParDE toxin-antitoxin system are present in the Vibrio cholerae genome within the superintegron on chromosome II. Here we found that these three loci-two of which have identical open reading frames and regulatory sequences-encode functional toxin-antitoxin systems. The ParE toxins inhibit bacterial division and reduce viability, presumably due to their capacity to damage DNA. The in vivo effects of ParE1/3 mimic those of ParE2, which we have previously demonstrated to be a DNA gyrase inhibitor in vitro, suggesting that ParE1/3 is likewise a gyrase inhibitor, despite its relatively low degree of sequence identity. ParE-mediated DNA damage activates the V. cholerae SOS response, which in turn likely accounts for ParE's inhibition of cell division. Each toxin's effects can be prevented by the expression of its cognate ParD antitoxin, which acts in a toxin-specific fashion both to block toxicity and to repress the expression of its parDE operon. Derepression of ParE activity in ΔparAB2 mutant V. cholerae cells that have lost chromosome II contributes to the prominent DNA degradation that accompanies the death of these cells. Overall, our findings suggest that the ParE toxins lead to the postsegregational killing of cells missing chromosome II in a manner that closely mimics postsegregational killing mediated by plasmid-encoded homologs. Thus, the parDE loci aid in the maintenance of the integrity of the V. cholerae superintegron and in ensuring the inheritance of chromosome II.     

The bacterial chromosome

Bacteria represent the vast majority of biological diversity found on Earth. In this review, we focus on selected aspects of their genetic material, those providing insight into structural, functional, dynamic, and evolutionary aspects of their genomes. Bacterial chromosomes are far more dynamic than previously realized, and dozens of mechanisms giving rise to genomic plasticity are now understood. Maturation of the genomics era has provided the tools for unraveling the interwoven details of DNA structure/function relationships that provide a basis for organismal diversity. Some of the most throughly understood processes that underlie the dynamics of genomic structure and function in prokaryotes are examined.     

Comparative chromosome painting

The review of the data on comparative chromosomal painting in mammals is presented. The development of new molecular-cytogenetic methods has resulted in the accumulation of the detailed information on homology of chromosomal segments of more than 50 species from 11 orders. In this review, modern methods of obtaining painting probes are considered in detail, and the basic tendencies of karyotype evolution in different taxa are discussed. Putative karyotypes of the ancestors of primates, carnivores, and placental mammals are considered.     

Counterstain-enhanced chromosome banding

Summary Chromosome staining, in which at least one member of a pair or triplet of DNA binding dyes is fluoescent whereas the others act as counterstain, is reviewed. Appropriately chosen combinations of fluorescent dyes and counterstains can be employed to enhance general chromosome banding patterns, or to induce specific regional banding patterns. Some pairs of dyes which exhibit complementary DNA binding specificity, A-T/G-C or G-C/A-T, provide enhanced definition of positive or reverse banding patterns. Dye combinations of the type A-T/A-T, that include two DNA stains with similar specificity but non-identical binding modes, produce a specific pattern of brightly fluorescnet heterochromatic regions (DA-DAPI bands). In man, the method highlights the C bands of chromosomes 1, 9, 15, 16, and the Y. Certain dye triplets of the type G-C/A-T/A-T, which include two spectroscopically separated fluorescent stains with reciprocal DNA base pair binding specificites and a non-fluorescent A-T binding counterstain, can be used to highlight selectively, in the appropriate wavelength ranges, either R bands or DA-DAPI bands.Applications of these techniques in human cytogenetics are described. The potential of the new methodology for detecting and analysing specific chromosome bands is demonstrated. The mechanisms responsible for contrast enhancement and pattern induction are reviewed and their implications for chromosome structure are discussed as they relate to the banding phenomenon and to the DNA composition of chromosomes.     

Meiosis and chromosome painting of sex chromosome systems in Ceboidea

The identity of the chromosomes involved in the multiple sex system of Alouatta caraya (Aca) and the possible distribution of this system among other Ceboidea were investigated by chromosome painting of mitotic cells from five species and by analysis of meiosis at pachytene in two species. The identity of the autosome #7 (X2) involved in the multiple system of Aca and its breakage points were demonstrated by both meiosis and chromosome painting. These features are identical to those described by Consigliere et al. [1996] in Alouatta seniculus sara (Assa) and Alouatta seniculus arctoidea (Asar). This multiple system was absent in the other four Ceboidea species studied here. However, data from the literature strongly suggest the presence of this multiple in other members of this genus. The presence of this multiple system among several species and subspecies that show high levels of chromosome rearrangements may suggest a special selective value of this multiple. The meiotic features of the sex systems of Aca and Cebus apella paraguayanus (Cap) are strikingly different at pachytene, as the latter system is similar to the sex pair of man and other primates. The relatively large genetic distances between species presently showing this multiple system suggest that its origin is not recent. Other members of the same genus should be investigated at meiosis and by chromosome painting in order to know the extent and distribution of this complex sex-chromosome system.     

Bacterial chromosome segregation

In most bacteria two vital processes of the cell cycle: DNA replication and chromosome segregation overlap temporally. The action of replication machinery in a fixed location in the cell leads to the duplication of oriC regions, their rapid separation to the opposite halves of the cell and the duplicated chromosomes gradually moving to the same locations prior to cell division. Numerous proteins are implicated in co-replicational DNA segregation and they will be characterized in this review. The proteins SeqA, SMC/MukB, MinCDE, MreB/Mbl, RacA, FtsK/SpoIIIE playing different roles in bacterial cells are also involved in chromosome segregation. The chromosomally encoded ParAB homologs of active partitioning proteins of low-copy number plasmids are also players, not always indispensable, in the segregation of bacterial chromosomes.     

Ring chromosome 16

Summary Ring chromosome 16 was found in a 33-year-old woman with mental, motor, and growth defects. Apart from a low percentage of monosomy 16 cell lines, the patient appears to have virtually all of the normal chromosome 16 genetic material at the microscopic level. Her impressive problems highlight the limitation of our ability to detect small deletions and the profound importance of the integrity of chromosome 16 in normal human development.     

Mitotic chromosome structure

Mounting evidence is compiling linking the physical organizational structure of chromosomes and the nuclear structure to biological function. At the base of the physical organizational structure of both is the concept of loop formation. This implies that physical proximity within chromosomes is provided for otherwise distal genomic regions and thus hierarchically organizing the chromosomes. Together with entropy many experimental observations can be explained with these two concepts. Among the observations that can be explained are the measured physical extent of the chromosomes, their shape, mechanical behavior, the segregation into territories (chromosomal and territories within chromosomes), the results from chromosome conformation capture experiments, as well as linking gene expression to structural organization.     

Mouse X chromosome

Overall, the probe map fromDXWas70 toAmg encompasses 72 cM and includes 103 loci. Eight of these have been designated reference loci (see Table 2 and previous section) on account of their wide usage that would enable the cross reference of independent maps created in different laboratories. Reference loci are to be readily available and easily used probes for Southern hybridization. By and large, they will be STSs, regeneratable by PCR, and correspond to a known gene. In addition, on the mouse X Chr, there is a reference locus from each of the known conserved linkage groups found between the mouse and human X Chrs. All the loci, barDXWas31, represent conserved sequences. Committee Members: D. Adler, P.J. Barnard, Y. Boyd, N. Brockdorff, J. Derry, C. Disteche, C. Faust, M.F. Lyon, S. Rastan, M. Seldin and L. Siracusa.     

TheAzotobacter vinelandii chromosome

The chromosome ofAzotobacter vinelandii was digested with the restriction endonucleasesSpeI (5’-ACTAGT),DraI (5’-TTTAAA) andAsel (5’-ATTAAT) and the products were separated by pulsed-field gel electrophoresis. The sum of the sizes of the restriction fragments comes to around 4.5 megabasepairs. Our earlier studies had revealed the presence of about 80 copies ofnifH, nifD, nifK andleuB genes in a log-phase cell ofA. vinelandii. To determine whether there are multiple identical chromosomes inA. vinelandii or one large chromosome with identical segments joined in tandem, we have subjected gamma-irradiated DNA ofA. vinelandii andEscherichia coli to pulsed-field gel electrophoresis. The results suggest thatA. vinelandii chromosomes contain multiple identical chromosomes of about the same size as that ofE. coli.     

Mouse chromosome 2

Chair of Committee for Mouse Chromosome 2     

Stylized chromosome images

Stylized chromosome images 1) serve as a format to test effects of preprocessing algorithms used in automated karyotyping; 2) enhance the ability of humans to perform quantitative analysis of chromosomal aberrations; 3) provide an alternative format for karyotype hard copies produced by automated systems. Stylized chromosomes are two-dimensional computer-generated images based on information extracted from one-dimensional width and density profiles. These profiles correspond to what cytogeneticists observe through the microscope as the shape and banding patterns of stained chromosomes. Stylized presentation sharpens chromosome band boundaries and perimeters, reduces "noise," and enhances gray level variations, which are difficult to distinguish by humans on photographic or computer generated karyotypes. Karyotyping accuracy using stylized images was used to detect difficult areas for automated chromosome identification. Landmark bands sufficient to classify chromosomes were identified; shapes of chromosomes reflected in width profiles were said to aid classification. A two-step automated karyotyping strategy proposed is: 1) classify chromosomes by landmarks, minimum information needed for identification; 2) subsequently employ the full banding pattern with maximum resolution to detect aberrations. Stylized images of abnormal chromosomes have potential for testing hypothesis regarding breakpoints and quantitative analysis, but improvements are needed in homologue normalization and definition of termini of chromosomes.     

Cross-species chromosome painting

Comparative genomics is an important and expanding field of research, and the genome-wide comparison of the chromosome constitution of different species makes a major contribution to this field. Cross-species chromosome painting is a powerful technique for establishing chromosome homology maps, defining the sites of chromosome fusions and fissions, investigating chromosome rearrangements during evolution and constructing ancestral karyotypes. Here the protocol for cross-species chromosome painting is presented. It includes sections on cell culture and metaphase preparation, labeling of chromosome-specific DNA, fluorescent in situ hybridization (chromosome painting) and image analysis. Cell culture and metaphase preparation can take between 1 and 2 wk depending on the cell culture. Labeling of chromosome-specific DNA is completed in 1 d. Fluorescent in situ hybridization can be completed in a maximum of 4 d.     

Human chromosome fragility

Fragile sites are heritable specific chromosome loci that exhibit an increased frequency of gaps, poor staining, constrictions or breaks when chromosomes are exposed to partial DNA replication inhibition. They constitute areas of chromatin that fail to compact during mitosis. They are classified as rare or common depending on their frequency within the population and are further subdivided on the basis of their specific induction chemistry into different groups differentiated as folate sensitive or non-folate sensitive rare fragile sites, and as aphidicolin, bromodeoxyuridine (BrdU) or 5-azacytidine inducible common fragile sites. Most of the known inducers of fragility share in common their potentiality to inhibit the elongation of DNA replication, particularly at fragile site loci. Seven folate sensitive (FRA10A, FRA11B, FRA12A, FRA16A, FRAXA, FRAXE and FRAXF) and two non-folate sensitive (FRA10B and FRA16B) fragile sites have been molecularly characterized. All have been found to represent expanded DNA repeat sequences resulting from a dynamic mutation involving the normally occurring polymorphic CCG/CGG trinucleotide repeats at the folate sensitive and AT-rich minisatellite repeats at the non-folate sensitive fragile sites. These expanded repeats were demonstrated, first, to have the potential, under certain conditions, to form stable secondary non-B DNA structures (intra-strand hairpins, slipped strand DNA or tetrahelical structures) and to present highly flexible repeat sequences, both conditions which are expected to affect the replication dynamics, and second, to decrease the efficiency of nucleosome assembly, resulting in decondensation defects seen as fragile sites. Thirteen aphidicolin inducible common fragile sites (FRA2G, FRA3B, FRA4F, FRA6E, FRA6F, FRA7E, FRA7G, FRA7H, FRA7I, FRA8C, FRA9E, FRA16D and FRAXB) have been characterized at a molecular level and found to represent relatively AT-rich DNA areas, but without any expanded repeat motifs. Analysis of structural characteristics of the DNA at some of these sites (FRA2G, FRA3B, FRA6F, FRA7E, FRA7G, FRA7H, FRA7I, FRA16D and FRAXB) showed that they contained more areas of high DNA torsional flexibility with more highly AT-dinucleotide-rich islands than neighbouring non-fragile regions. These islands were shown to have the potential to form secondary non-B DNA structures and to interfere with higher-order chromatin folding. Therefore, a common fragility mechanism, characterized by high flexibility and the potential to form secondary structures and interfere with nucleosome assembly, is shared by all the cloned classes of fragile sites. From the clinical point of view, the folate sensitive rare fragile site FRAXA is the most important fragile site as it is associated with the fragile X syndrome, the most common form of familial mental retardation, affecting about 1/4000 males and 1/6000 females. Mental retardation in this syndrome is considered as resulting from the abolition of the FMR1 gene expression due to hypermethylation of the gene CpG islands adjacent to the expanded methylated trinucleotide repeat. FRAXE is associated with X-linked non-specific mental retardation, and FRA11B with Jacobsen syndrome. There is also some evidence that fragile sites, especially common fragile sites, are consistently involved in the in vivo chromosomal rearrangements related to cancer, whereas the possible implication of common fragile sites in neuropsychiatric and developmental disorders is still poorly documented.     

Mouse chromosome 1

Committee Members: D. Epstein, T.A. Howard, D. Malo, F. Mancino, M. Mehrabian, K.J. Moore, R.J. Oakey, S. Reinehez-Videl, K. Steel and M.L. Watson.