Showing papers on "Chromosome published in 1988"

Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries

Abstract: A method of in situ hybridization for visualizing individual human chromosomes from pter to qter, both in metaphase spreads and interphase nuclei, is reported. DNA inserts from a single chromosomal library are labeled with biotin and partially preannealed with a titrated amount of total human genomic DNA prior to hybridization with cellular or chromosomal preparations. The cross-hybridization of repetitive sequences to nontargeted chromosomes can be markedly suppressed under appropriate preannealing conditions. The remaining single-stranded DNA is hybridized to specimens of interest and detected with fluorescent or enzymelabeled avidin conjugates following post-hybridization washes. DNA inserts from recombinant libraries for chromosomes 1, 4, 7, 8, 13, 14, 18, 20, 21, 22, and X were assessed for their ability to decorate specifically their cognate chromosome; most libraries proved to be highly specific. Quantitative densitometric analyses indicated that the ratio of specific to nonspecific hybridization signal under optimal preannealing conditions was at least 8:1. Interphase nuclei showed a cohesive territorial organization of chromosomal domains, and laserscanning confocal fluorescence microscopy was used to aid the 3-D visualization of these domains. This method should be useful for both karyotypic studies and for the analysis of chromosome topography in interphase cells.

RFLP Maps Based on a Common Set of Clones Reveal Modes of Chromosomal Evolution in Potato and Tomato.

Abstract: Potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum) are members of the Solanaceae (nightshade family) and have the same basic chromosome number (x = 12). However, they cannot be cross-hybridized and, until now, it was unknown how conserved the gene order might be between these two species. We report herein the construction of a genetic linkage map of potato chromosomes based on genomic and cDNA clones from tomato. The potato map was drawn from segregation data derived from the interspecific cross S. phureja X (S. tuberosum X S. chacoense) (2n = 2x = 24), and consists of 135 markers defining 12 distinct linkage groups. Nearly all of the tomato probes tested hybridized to potato DNA, and in most cases, the copy number of the employed clones was the same in both species. Furthermore, all clones mapped to the same linkage group in both species. For nine chromosomes, the order of loci appears to be identical in the two species, while for the other three, intrachromosomal rearrangements are apparent, all of which appear to be paracentric inversions with one breakpoint at or near the centromere. These results are consistent with cytogenetic theory, previously untested in plants, which predicts that paracentric inversions will have the least negative effect on fitness and thus be the most likely form of chromosomal rearrangements to survive through evolutionary time. Linkage maps based on a common set of restriction fragment length polymorphism markers provide a basis for uniting the previously separate disciplines of tomato and potato genetics. Using these maps, it may now be possible to test theories about homologies or orthologies of other genes, including those coding for disease resistance and stress tolerances.

Uniparental disomy as a mechanism for human genetic disease.

Abstract: A female with cystic fibrosis and short stature was investigated for molecular or cytogenetic abnormalities that might explain the combined phenotype. Analysis with polymorphic DNA markers indicated that the father did not contribute alleles to the propositus for markers near the CF locus or for centromeric markers on chromosome 7. High-resolution cytogenetic analysis was normal, and the result could not be explained on the basis of nonpaternity or a submicroscopic deletion. All of the data indicate that the propositus inherited two identical copies of maternal sequences for much or all of chromosome 7. The occurrence of uniparental disomy could be explained by models postulating postfertilization error, gamete complementation, monosomic conception with subsequent chromosome gain, or trisomic conception followed by chromosome loss. Uniparental disomy in an individual with a normal chromosome analysis is a novel mechanism for the occurrence of human genetic disease.

Rapid detection of human chromosome 21 aberrations by in situ hybridization

Abstract: Plasmid clones containing up to 94 kilobases of single-copy DNA from band q22.3 of chromosome 21 and a complete pool of insert DNA from a chromosome 21 recombinant library have been used to rapidly detect numerical and structural aberrations of chromosome 21 by in situ hybridization in both metaphase and interphase cells. A trisomic karyotype, diagnostic of Down syndrome, is readily detected in nonmitotic cells because the majority of their nuclei exhibit three discrete foci of hybridization, in contrast to normal diploid cells, which show two foci. Chromosomal translocations involving chromosome 21 sequences were also detected with these probes, and the intranuclear location of 21q22.3 DNA sequences in "normal" human brain neurons was established with the plasmid DNA probe set. These results suggest that chromosome 21-specific probes may have utility in clinical diagnostics, especially by facilitating the direct analysis of interphase cells.

Detection of Chromosome Aneuploidy in Interphase Nuclei from Human Primary Breast Tumors Using Chromosome-specific Repetitive DNA Probes

Abstract: We have used in situ hybridization with chromosome specific repetitive DNA sequences as a probe to reveal particular chromosomes as distinct spots or clusters of signal within interphase nuclei. Using karyotypically defined cells and cell lines, we show that the number of signals obtained per nucleus correlates with the number of particular chromosomes present in that nucleus. Further, admixtures of karyotypically different cell lines could be detected. In situ hybridization of nuclei and metaphase spreads derived from the breast cancer cell line MCF-7 shows that a deviant number of spots/nucleus indicates a numerical and/or structural chromosomal aberration. In seven primary breast tumors studied, we detected numerical aberrations of the target sites of chromosomes 1 and/or 18. Although all had a single peak in DNA flow measurements, six of the cases appeared to be heterogeneous with respect to their spots/nucleus content.

Different regions of the immunoglobulin heavy-chain locus are involved in chromosomal translocations in distinct pathogenetic forms of Burkitt lymphoma.

Abstract: We show that endemic (eBL), sporadic (sBL), and acquired immunodeficiency syndrome-associated (AIDS-BL) forms of Burkitt lymphoma (BL) carrying t(8;14) chromosomal translocations display different breakpoints within the immunoglobulin heavy-chain locus (IGH) on chromosome 14 In sBL (7 out of 11) and AIDS-BL (5 out of 6), the breakpoints occurred within or near the IGH mu switch (S-mu) region on chromosome 14 and within the c-myc locus (MYC) on chromosome 8 In most eBL (13 out of 16) the breakpoints were mapped within or 5' to the IGH joining (JH region on chromosome 14 and outside the MYC locus on chromosome 8 Cloning and sequencing of the t(8;14) chromosomal junctions from two eBL cell lines and one eBL biopsy sample show that the recombinations do not involve IGH-specific recombination signals on chromosome 14 or homologous sequences on chromosome 8, suggesting that these events are not likely to be mediated by the same mechanisms or enzymes as in IGH rearrangements In general, these data have implications for the timing of occurrence of chromosomal translocations during B-cell differentiation in different BL types

The short arm of chromosome 11 is a "hot spot" for hypermethylation in human neoplasia.

Abstract: Inactivation of normally expressed genes may play a role in the formation and/or progression of human cancers. Methylation of cytosine in DNA could potentially participate in such alterations of gene expression. Abnormalities in DNA methylation are a consistent feature of human neoplasms, and we now show that these include not only previously recognized widespread genomic hypomethylation, but also regional increases in gene methylation. A hot spot for abnormal methylation of C + G-rich areas has been detected on the short arm of chromosome 11 in an area known to harbor tumor suppressor genes. This change occurs consistently in common forms of human cancer and appears early during the transformation of cells with viruses including members of the human T-cell leukemia (HTLV) family. Furthermore, in one chromosome 11 gene examined, calcitonin, the increased methylation in somatic tumor cells coincides with the presence of an "inactive" chromatin pattern in the transcriptional regulatory area. The increased regional DNA methylation demonstrated may then participate in or mark chromosomal changes associated with gene inactivation events that are central to the genesis and/or progression of human cancers.

Chromosomal reorganization for the expression of recessive mutation of retinoblastoma susceptibility gene in the development of osteosarcoma.

Abstract: Recent evidence indicates that the mutation of retinoblastoma susceptibility (RB) gene is also involved in the development of osteosarcoma. We studied 30 cases of osteosarcoma for the structural anomalies of the RB gene by Southern hybridization analysis with cDNA probes of the RB gene. Thirteen cases (43%) showed structural anomalies of the RB gene. They included the total or partial deletion, or rearrangement of the RB gene; seven with homozygous deletions and six with hemizygous deletions or rearrangements. By the use of restriction fragment length polymorphism fragments as chromosome markers, those seven tumors having homozygous deletions and four of six tumors having hemizygous anomalies showed the loss of heterozygosity at other loci on chromosome 13. Among those tumors with no apparent structural changes of the RB gene, seven cases showed the loss of heterozygosity on chromosome 13, and altogether the loss of heterozygosity by either homozygosity or hemizygosity was found in 18 (64%) of 28 informative cases. The loss of heterozygosity was also found for nine of 10 other chromosomes, of which chromosome 17 showed the highest frequency (77%). The tumors with loss of chromosome 13 alleles also showed additional losses of alleles on other chromosomes, while tumors retaining heterozygosity of chromosome 13 also retained heterozygosity at the informative loci on other chromosomes. Southern hybridization and karyotype analysis in some selected cases suggest that the concerted loss of heterozygosity at multiple loci may be a consequence of the polyploidization-segregation process.

Evolutionary diversity of reverse (R) fluorescent chromosome bands in vertebrates

Abstract: Mitotic chromosomes, interphase cell nuclei, and male meiosis of 41 species representing all vertebrate classes were analyzed with distamycin A/mithramycin counterstaining. The purpose of the study was to recognize differences and common characteristics in the reverse (R) fluorescent banding patterns in the chromosomes of vertebrate species at various stages of evolution. In contrast to the warm-blooded mammals and birds, the euchromatic segments in the chromosomes of most reptiles, amphibians, and fishes contain no multiple fluorescent R-bands. This is thought to be due to the absence of the long homogeneous regions (isochores) in the DNA of the cold-blooded vertebrates. Distamycin A/mithramycin banding specifically reveals the GC-rich constitutive heterochromatin in all vertebrates. In most of the vertebrate chromosomes examined, the heterochromatic regions have opposite staining properties with mithramycin and quinacrine. Mithramycin labels the nucleolus organizer regions very brightly in the karyotypes of fishes, amphibians, reptiles and birds, but not of mammals. The lack of mithramycin fluorescence at the nucleolus organizer regions of mammals is attributed to the relatively low level of redundancy of the GC-rich ribosomal DNA in their genomes. Studies on the various meiotic stages of the cold-blooded vertebrates show that the mithramycin labeling of the nucleolus organizers is independent of their state of activity. This can be confirmed by mithramycin fluorescence at the nucleoli of actinomycintreated cells.

Telomeric repeat from T. thermophila cross hybridizes with human telomeres

Abstract: The ends (telomeres) of eukaryotic chromosomes must have special features to ensure their stability and complete replication. Studies in yeast, protozoa, slime moulds and flagellates show that telomeres are tandem repeats of simple sequences that have a G-rich and a C-rich strand. Mammalian telomeres have yet to be isolated and characterized, although a DNA fragment within 20 kilobases of the telomeres of the short arms of the human sex chromosomes has been isolated. Recently we showed that a chromosome from the fission yeast Schizosaccharomyces pombe could, in some cases, replicate as an autonomous mini-chromosome in mouse cells. By extrapolation from other systems, we reasoned that mouse telomeres could be added to the S. pombe chromosome ends in the mouse cells. On setting out to test this hypothesis we found to our surprise that the telomeric probe used (containing both the S. pombe and Tetrahymena thermophila repeats) hybridized to a series of discrete fragments in normal mouse DNA and DNA from a wide range of eukaryotes. We show here that the sequences hybridizing to this probe are located at the telomeres of most, if not all, human chromosomes and are similar to the Tetrahymena telomeric-repeat component of the probe.

A "selfish" B chromosome that enhances its transmission by eliminating the paternal genome.

Abstract: In the parasitic wasp, Nasonia vitripennis, males are haploid and usually develop from unfertilized eggs, whereas females are diploid and develop from fertilized eggs. Some individuals in this species carry a genetic element, termed psr (paternal sex ratio), which is transmitted through sperm and causes condensation and subsequent loss of paternal chromosomes in fertilized eggs, thus converting diploid females into haploid males. In this report the psr trait was shown to be caused by a supernumerary chromosome. This B chromosome contains at least three repetitive DNA sequences that do not cross-hybridize to each other or to the host genome. The psr chromosome apparently produces a trans-acting product responsible for condensation of the paternal chromosomes, but is itself insensitive to the effect. Because the psr chromosome enhances its transmission by eliminating the rest of the genome, it can be considered the most "selfish" genetic element yet described.

Induction of Chromosomal Structural Changes by a Chromosome of Aegilops cylindrica L. in Common Wheat

Abstract: Chromosomal structural changes-deletions and translocations-occurred in almost half the progeny of a monosomic addition line of common wheat, Triticum aestivum (2n = 42, AABBDD), which had a chromosome from Aegilops cylindrica (2n = 28, CCDD). Most of the progeny with chromosomal structural changes lacked the A. cylindrica chromosome. Chromosome breaks were observed in various regions of all the wheat chromosomes and the A. cylindrica chromosome. Chromosome aberrations occurred far less frequently both in the self-progeny of the disomic addition plants and in the F1 monosomic addition plants derived from reciprocal crosses with normal common wheat. These findings suggest that when the A. cylindrica chromosome was in the sporophytes, chromosome breakage was likely to occur in the gametophytes that lacked it but that the gametophytes were still functional, resulting in the production of offspring with chromosomal structural changes.

Physical mapping of large DNA by chromosome fragmentation.

Abstract: A technique is described for physically positioning any cloned DNA on a native or artificial Saccharomyces cerevisiae chromosome. The technique involves splitting a chromosome at a specific site by transformation with short linear molecules containing the cloned DNA at one end and telomeric sequences at the other. Recombination between the end of the linear molecules and homologous chromosomal sequences gives rise to chromosome fragments comprising all sequences distal or proximal to the mapping site depending on the orientation of the cloned DNA. The recombinant products are recovered by screening for stabilization of a suppressor tRNA on the linear molecules using a colony color assay. The cloned DNA is positioned relative to the chromosome ends by sizing the chromosomal fragments using alternating contour-clamped homogeneous electric field gel electrophoresis. Application of this technique to organisms other than S. cerevisiae and to the analysis of exogenous DNA cloned in yeast is discussed.

Transcription interferes with elements important for chromosome maintenance in Saccharomyces cerevisiae.

Abstract: Transcription directed into a Saccharomyces cerevisiae autonomously replicating sequence (ARS) causes high-frequency loss of minichromosomes. Conditionally stable artificial yeast chromosomes were constructed that contain an inducible GAL promoter upstream of ARS1. Under growth conditions in which the promoter was inactive, these chromosomes were mitotically stable; however, when the GAL promoter was induced, the chromosomes became extremely unstable as a result of transcriptional impairment of ARS function. This interference by the GAL promoter occurred only in cis but can occur from either side of ARS1. Transcriptional interference of ARS function can be monitored readily by using a visual colony-color assay (P. Hieter, C. Mann, M. Snyder, and R.W. Davis, Cell 40:381-392, 1985), which was further developed as a sensitive in vivo assay for sequences which rescue ARS from transcription. DNA fragments from the 3' ends of genes, inserted downstream of the GAL promoter, protected ARS function from transcriptional interference. This assay is expected to be independent of both RNA transcript stability and processing. Philippsen et al. have shown that transcription into a yeast centromere inhibits CEN function in vivo (L. Panzeri, I. Groth-Clausen, J. Shepard, A. Stotz, and P. Philippsen, Chromosomes Today 8:46-58, 1984). We identified two 200- to 300-base-pair DNA fragments flanking CEN4 that rescued ARS1 from transcription. Both of these fragments protected ARS from transcription when inserted in either orientation. The 3' ends of stable transcripts are encoded by fragments that protected the ARS from transcription, suggesting that the protection was achieved by transcription termination. It is suggested that protection of elements important for the replication and segregation of eucaryotic chromosomes from transcription is necessary for their proper function in vivo.

Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae

Abstract: Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.

Polytene chromosomes in mouse trophoblast giant cells

Abstract: Mouse trophoblast giant cells undergo successive rounds of DNA replication resulting in amplification of the genome. It has been difficult to determine whether giant cell chromosomes are polyploid as in liver cells or polytene as in Dipteran salivary glands because the chromosomes do not condense. We have examined the pattern of hybridization of mouse giant cells with a variety of in situ chromosome markers to address this question. Hemizygous markers displayed one hybridization signal per nucleus in both diploid and giant cells, while homozygous markers displayed two signals per nucleus in both cell types. These patterns are consistent with cytological evidence indicating that giant cell chromosomes are polytene rather than polyploid. However, in contrast to the situation in Dipteran salivary glands, the two homologues do not appear to be closely associated. We conclude that the mechanism of giant cell DNA amplification involves multiple rounds of DNA replication in the absence of both karyokinesis and cytokinesis, and that sister chromatids, but not homologous chromosomes, remain closely associated during this process.

Close association of a DNA replication origin and an ARS element on chromosome III of the yeast, Saccharomyces cerevisiae.

Abstract: Two dimensional gel electrophoretic techniques were used to locate all functional DNA replication origins in a 22.5 kb stretch of yeast chromosome III. Only one origin was detected, and that origin is located within several hundred bp of an ARS element.

The genomic relationship between Glycine max (L.) Merr. and G. soja Sieb. and Zucc. as revealed by pachytene chromosome analysis.

Abstract: This study was conducted with the objective of determining the genomic relationship between cultivated soybean (Glycine max) and wild soybean (G. soja) of the subgenus Soja, genus Glycine. Observations on cross-ability rate, hybrid viability, meiotic chromosome pairing, and pollen fertility in F 1 hybrids of G. max × G. soja and reciprocals elucidated that both species hybridized readily and set mature putative hybrid pods, generated vigorous F1 plants, had a majority of sporocytes that showed 18II + 1IV chromosome association at diakinesis and metaphase I, and had a pollen fertility that ranged from 49.2% to 53.3%. A quadrivalent was often associated with the nucleolus, suggesting that one of the chromosomes involved in the interchange is a satellited chromosome. Thus, G. max and G. soja genetic stocks used in this study have been differentiated by a reciprocal translocation. Pachytene analysis of F1 hybrids helped construct chromosome maps based on chromosome length and euchromatin and heterochromatin distribution. Chromosomes were numbered in descending order of 1–20. Pachytene chromosomes in soybean showed heterochromatin distribution on either side of the centromeres. Pachytene analysis revealed small structural differences for chromosomes 6 and 11 which were not detected at diakinesis and metaphase I. This study suggests that G. max and G. soja carry similar genomes and validates the previously assigned genome symbol GG.

Klinefelter's syndrome: an analysis of the origin of the additional sex chromosome using molecular probes.

Abstract: The results of our study of the origin of the additional X chromosome in 39 males with a 47,XXY chromosome constitution are reported. We used a total of 20 X-linked RFLPs and successfully determined the origin of all 32 patients in whom DNA from both parents was available, and a further 3 in whom DNA was available from the patient and mother only. Males whose additional X chromosome was maternal in origin were further investigated using an X-linked centromere specific probe to determine the cell division at which the error occurred. Our results showed 53% of the non-disjunction to be attributable to pat mei I errors, 34% to mat mei I errors, 9% to mat mei II errors and 3% to a post-zygotic mitotic error. In the great majority of patients resulting from an error of maternal meiosis there was clear evidence of recombination involving the non-disjoined chromosomes, suggesting that absence of recombination is not an important aetiological factor in non-disjunction of the X chromosome in female meiosis. There was no alteration of parental age associated with the paternally derived 47,XXY males but a marked increase in maternal age among the maternally derived 47,XXY males, the increase being associated with mat mei I but not mat mei II errors. The proportion of paternally and maternally derived cases was similar among different ascertainment classes, suggesting that there is no dramatic effect of parental origin of the additional X chromosome on the phenotype of 47,XXY males.

Assignment of cloned genes to the seven electrophoretically separated Candida albicans chromosomes.

Abstract: By using orthogonal-field alternating gel electrophoresis (OFAGE), field-inversion gel electrophoresis (FIGE), and contour-clamped homogeneous field gel electrophoresis (CHEF), we have clearly resolved 11 chromosomal bands from various Candida albicans strains. OFAGE resolves the smaller chromosomes better, while FIGE, which under our conditions causes the chromosomes to run in the reverse order of OFAGE, is more effective in separating the larger chromosomes. CHEF separates all chromosomes under some conditions, but these conditions do not often resolve homologs. The strains examined are highly polymorphic for chromosome size. Fourteen cloned Candida genes, isolated on the basis of conferral of new properties to or complementation of auxotrophic deficiencies in Saccharomyces cerevisiae, and three sequences of unknown function have been hybridized to Southern transfers of CHEF, FIGE, and OFAGE gels. Four sets of resolvable bands have been shown to be homologous chromosomes. On the basis of these data, we suggest that C. albicans has seven chromosomes. Genes have been assigned to the seven chromosomes. Two chromosomes identified genetically have been located on the electrophoretic karyotype.

RED1: a yeast gene required for the segregation of chromosomes during the reductional division of meiosis

Abstract: A mutation at the RED1 locus was identified in a search for sporulation-proficient, meiotic-lethal yeast mutants. The few viable spores produced in the red1-1 mutant are highly aneuploid, suggesting that the spore lethality results from a high frequency of chromosome nondisjunction. Disomic spores produced by the red1-1 mutant contain nonsister chromatids and the red1-1 spore inviability phenotype is alleviated in red1-1 spo13 double mutants; these results indicate that nondisjunction occurs at the first meiotic division. The red1-1 mutant is recombination-proficient. The RED1 gene was cloned by complementation of the meiotic lethal phenotype; strains carrying a disruption of the gene are mitotically viable. We propose that the RED1 gene product is involved in meiosis I chromosome disjunction, perhaps by maintaining the connections between homologous chromosomes through metaphase I.