Non-random distribution of Alu-family repeats in human chromosomes

A phage lambda recombinant clone containing at least 8 Alu-family repeats (AFRs) has been isolated from a human genomic library, and DNA from the phage was used as a probe for in situ hybridization on G-banded human metaphase chromosomes of healthy donors and leukemic patients. Some chromosome bands show prominent clusters of silver grains in all individuals examined: 1p34, 1q23, 2q21–22, 10p14, 11p14, 10q21 and 11q14. The data suggest non-random distribution of AFRs in the human genome.     

The chromosomal distribution of microsatellite repeats in the genome of the wolf fish Hoplias malabaricus, focusing on the sex chromosomes

Distribution of 12 mono-, di- and tri-nucleotide microsatellites on the chromosomes of 2 karyomorphs with 2 distinct sex chromosome systems (a simple XX/XY - karyomorph B and a multiple X(1)X(1)X(2)X(2)/X(1)X(2)Y - karyomorph D) in Hoplias malabaricus, commonly referred to as wolf fish, was studied using their physical mapping with fluorescence in situ hybridization (FISH). The distribution patterns of different microsatellites along the chromosomes varied considerably. Strong hybridization signals were observed at subtelomeric and heterochromatic regions of several autosomes, with a different accumulation on the sex chromosomes. A massive accumulation was found in the heterochromatic region of the X chromosome of karyomorph B, whereas microsatellites were gathered at centromeres of both X chromosomes as well as in corresponding regions of the neo-Y chromosome in karyomorph D. Our findings are likely in agreement with models that predict the accumulation of repetitive DNA sequences in regions with very low recombination. This process is however in contrast with what was observed in multiple systems, where such a reduction might be facilitated by the chromosomal rearrangements that are directly associated with the origin of these systems.     

Telomere repeats within the neo-Y-chromosome of Drosophila miranda

The clone Dmir1098, isolated from a genomic lambda library of Drosophila miranda labels exclusively the tips of the giant chromosomes in the highly polytenized nuclei of the female larval salivary glands. However, the in situ hybridizations to male metaphase plates, using the same probe, reveal a massive labeling block within the neo-Y-chromosome in addition to the labeling blocks at both chromosome ends. From the comparison with the Y chromosome labeling pattern of D. pseudoobscura, a sibling species to D. miranda, an end-to-end fusion mechanism involving the telomere repeats would be the most straightforward explanation for the karyotype change in D. miranda.     

Differential distribution of RNA polymerase B and nonhistone chromosomal proteins in polytene chromosomes of Drosophila melanogaster

The distribution patterns of nonhistone chromosomal proteins (NHCP) associated with pulse-labeled RNA were determined by indirect immunofluorescence on salivary gland chromosomes of Drosophila melanogaster using monoclonal antibodies. By staining for two different antigens simultaneously, using antibodies tagged with different fluorescent probes, it became possible to position RNA-associated antigens as well as RNA polymerase B in relation to each other. Three separate staining patterns could be observed with anti-NHCP antibodies, none of which showed a pattern which was identical with that of RNA polymerase B. Furthermore, no correlation with the synthesis of the primary trancript, as monitored by the RNA polymerase B content of chromosomal sites, could be found by following the fluorescence patterns during inactivation of intermolt puffs or activation of early ecdysone-induced puffs. Finally, no strict correlation was observed between puffing activity and the accumulation of a certain antigen in these selected chromosomal sites.     

The distribution of two highly repeated DNA sequences within Drosophila melanogaster chromosomes

In situ hybridization using ³H-RNA probes has been used to localize the sequences found in two satellites of density 1.705 g/cc and 1.672 g/ cc to specific sites within the chromosomal complement. A detailed analysis of the sites on the X chromosome was carried out using the scute series of inversions to relate the heterochromatic breakpoint relative to the location of the sequence on this chromosome. It has also been possible to establish the order of arrangement of 1.705 and 1.672 DNA at the heterochromaticeuchromatic junction on chromosome 3(R). A mitotic map is provided. The T_m of hybrids formed in situ showed that the hybrids were representative of the sequences being analyzed. The two satellites also were traced through a number of purification procedures to show that a covalent linkage may be likely between the 1.705 g/cc and 1.672 g/cc satellite as predicted from in situ hybridization analyses.     

The distribution of transposable elements within and between chromosomes in a population of Drosophila melanogaster. I. Element frequencies and distribution.

Data were collected on the distribution of nine families of transposable elements among second and third chromosomes isolated from a natural population of Drosophila melanogaster, by means of in situ hybridization of element probes to polytene chromosomes. It was found that the copy numbers per chromosome in the distal sections of the chromosome arms followed a Poisson distribution. Elements appeared to be distributed randomly along the distal sections of the chromosome arms. There was no evidence for linkage disequilibrium in the distal sections of the chromosomes, but some significant disequilibrium was detected in proximal regions. There were many significant correlations between different element families with respect to the identity of the sites that were occupied in the sample. There were also significant correlations between families with respect to sites at which elements achieved relatively high frequencies. Element frequencies per chromosome band were generally low in the distal sections, but were higher proximally. These results are discussed in the light of models of the population dynamics of transposable elements. It is concluded that they provide strong evidence for the operation of a force or forces opposing transpositional increase in copy number. The data suggest that the rate of transposition per element per generation is of the order of 10(-4), for the elements included in this study.     

Similarities in chromosomal puffing induced by temperature shocks and dinitrophenol in Drosophila

A comparison between chromosomal puffs induced by temperature shocks and dinitrophenol in regions 87 A and 87 B of salivary gland chromosome 3 R of Drosophila melanogaster is presented. The size of both puffs can be regulated by either the temperature of a heat-shock or by the concentration of dinitrophenol. The two agents are additive with respect to their effects upon puff size, supporting the idea that both result in a change in ATP synthesis within the cell. A possible relationship between these agents and puff formation and RNA metabolism is discussed.This investigation was supported by research grant No. 119-70 from the Cancer Association of Greater New Orleans.     

Dinucleotide repeats in the Drosophila and human genomes have complex,length-dependent mutation processes

We use methods of maximum likelihood estimation to fit several microsatellite mutation models to the observed length distribution of dinucletoide repeats in the Drosophila and human genomes. All simple models are rejected by this procedure. Two new models, one with quadratic and another with piecewise linear slippage rates, have the best fits and agree with recent experimental studies by predicting that long microsatellites have a bias toward contractions.     

Localization of chromosomal RNA in human G-banded metaphase chromosomes

Chromosomal RNA with an 11% dihydropyrimidine content was extracted from human placental chromatin. Under appropriate conditions, this RNA showed wide-spread in situ hybridization to metaphase chromosomes. This included preferential hybridization to the telomeric regions and heterochromatic short arms of acrocentric chromosomes as well as significant hybridization to Q- and G-positive bands.     

Mechanisms of genetic exchange within the chromosomal inversions of Drosophila pseudoobscura

We have used the inversion system of Drosophila pseudoobscura to investigate how genetic flux occurs among the gene arrangements. The patterns of nucleotide polymorphism at seven loci were used to infer gene conversion events between pairs of different gene arrangements. We estimate that the average gene conversion tract length is 205 bp and that the average conversion rate is 3.4 x 10(-6), which is 2 orders of magnitude greater than the mutation rate. We did not detect gene conversion events between all combinations of gene arrangements even though there was sufficient nucleotide variation for detection and sufficient opportunity for exchanges to occur. Genetic flux across the inverted chromosome resulted in higher levels of differentiation within 0.1 Mb of inversion breakpoints, but a slightly lower level of differentiation in central inverted regions. No gene conversion events were detected within 17 kb of an inversion breakpoint suggesting that the formation of double-strand breaks is reduced near rearrangement breakpoints in heterozygotes. At least one case where selection rather than proximity to an inversion breakpoint is responsible for reduction in polymorphism was identified.     

Functional gene groups are concentrated within chromosomes,among chromosomes and in the nuclear space of the human genome

Genomes undergo changes in organization as a result of gene duplications, chromosomal rearrangements and local mutations, among other mechanisms. In contrast to prokaryotes, in which genes of a common function are often organized in operons and reside contiguously along the genome, most eukaryotes show much weaker clustering of genes by function, except for few concrete functional groups. We set out to check systematically if there is a relation between gene function and gene organization in the human genome. We test this question for three types of functional groups: pairs of interacting proteins, complexes and pathways. We find a significant concentration of functional groups both in terms of their distance within the same chromosome and in terms of their dispersal over several chromosomes. Moreover, using Hi-C contact map of the tendency of chromosomal segments to appear close in the 3D space of the nucleus, we show that members of the same functional group that reside on distinct chromosomes tend to co-localize in space. The result holds for all three types of functional groups that we tested. Hence, the human genome shows substantial concentration of functional groups within chromosomes and across chromosomes in space.     

Conversion and reciprocal exchange between tandem repeats in Drosophila melanogaster

Summary We have developed an experimental system to assay conversion and reciprocal exchange between tandem repeats in Drosophila melanogaster. In this system, the recombining markers map 0.76 kb apart within the Adh gene, and the length of the repeated unit is 4.75 kb. Our results provide a preliminary record of germline frequencies of gene conversion and unequal exchange between these markers. Conversions involving dispersed repeats were not observed, and may be less frequent. This work demonstrates that conversion takes place at an appreciable frequency between tandem repeats in metazoan germline. It confirms that gene conversion can mediate homogenization of reiterated sequences in higher eukaryotes.     

Functional genomics of histone modification and non-histone chromosomal proteins using the polytene chromosomes of Drosophila

Knowing the genomic distribution of chromosomal proteins and of histone modifications provides essential insight into function. The giant polytene chromosomes of the Drosophila larval salivary glands provide a high-resolution genomic map with a resolution exceeded only by chromatin immunoprecipitation. Immunofluorescence localization of chromosomal proteins and specific post-translational modifications of histones is a simple and rapid tool for the functional genomics of chromosomal proteins.     

The distribution of transposable elements within and between chromosomes in a population of Drosophila melanogaster. II. Inferences on the nature of selection against elements.

Data were collected on the distribution of nine families of transposable elements among a sample of autosomes isolated from a natural population of Drosophila melanogaster, by means of in situ hybridization of biotinylated probes to polytene chromosomes. There is no general tendency for elements to accumulate at the tips of chromosomes. Elements tend to be present in excess of random expectation in the euchromatin proximal to the centromeres of the major autosomes, and on chromosome four. There is considerable heterogeneity between different families in the extent of this excess. The overall abundance of element families is inversely related to the extent to which they accumulate proximally. The level of proximal accumulation for the major autosomes is similar to that on the fourth chromosome, but less than that for the X chromosome. There is an overall deficiency of elements in the mid-section of the X compared with the mid-sections of the major autosomes, with considerable heterogeneity between families. The magnitude of this deficiency is positively related to the extent to which elements accumulate proximally. No such deficiency is seen if the proximal regions of the X and autosomes are compared. There is a small and non-significant excess of elements in third chromosomes carrying inversions. There is some between-year heterogeneity in element abundance. The implications of these findings are discussed, and it is concluded that they generally support the hypothesis that transposable element abundance is regulated primarily by the deleterious fitness consequences of meiotic ectopic exchange between elements. If this is the case, such exchange must be very infrequent in the proximal euchromatin, and the elements detected in population surveys of this kind must be inserted into sites where they have negligible mutational effects on fitness.     

Healing of broken human chromosomes by the addition of telomeric repeats.

We have characterized and compared a series of naturally occurring chromosomal truncations involving the terminal region of the short arm of human chromosome 16 (16p13.3). All six broken chromosomes appear to have been stabilized by the direct addition of telomeric repeats (TTAGGG)n to nontelomeric DNA. In five of the six chromosomes, sequence analysis shows that the three of four nucleotides preceding the point of telomere addition are complementary to and in phase with the putative RNA template of human telomerase. Otherwise we have found no common structural features around the breakpoint regions. These findings, together with previously reported in vitro data, suggest that chromosome-healing events in man can be mediated by telomerase and that a small region of complementarity to the RNA template of telomerase at the end of a broken chromosome may be sufficient to prime healing in vivo.     

An in situ study of variant telomeric repeats in human chromosomes.

Variant telomeric repeats are selectively detected in human telomeres in situ by the novel approach of dideoxy-PRINS, displaying their organization in a format where all the individual chromosome ends can be viewed individually and simultaneously. All human chromosome ends are found to contain variant repeats, though not all types of repeats can be detected on all chromosome ends. Although the staining frequency at particular chromosome ends seems polymorphic among individuals, some chromosome ends are more commonly stained with a given probe than others. A few chromosome ends also appear with particularly strong signals. With a probe for one type of variant repeat ((AGGGTG)n), peculiar patterns with more than two signals per chromosome end are observed.     

Differential chromosomal distribution of ribonucleoprotein antigens in nuclei of Drosophila spermatocytes

The ribonucleoprotein (RNP) composition of the active Y chromosomal structures in spermatocyte nuclei of Drosophila hydei has been investigated using the anti-RNP antibodies Dm 28K2 and pp60 as a probe. Antibody Dm 28K2 was raised against an RNP protein of cytoplasmic RNP particles in D. melanogaster cells, while antibody pp60 was raised against a pre-messenger RNP fraction from oocytes of Xenopus laevis. Both antibodies detect nuclear RNP (nRNP) antigens of D. hydei. This is shown by CsCl density centrifugation of nRNP from D. hydei cells and immunoblotting across the density gradient. Dm 28K2 and pp60 recognize antigens of nRNP complexes which band at a characteristic buoyant density of approximately 1.4 g/cm3 in CsCl. By indirect immunofluorescence we observe that the nRNP complexes identified by Dm 28K2 are localized at only two of the five Y chromosomal loop structures which are named according to their distinct morphology. Dm 28K2 decorates RNPs within the "clubs," within the cones, and within the matrix of the "pseudonucleolus." Ultrastructural bodies that are candidates for this immunoreaction are RNP granules that resemble the so-called perichromatin granules. Antibody pp60 recognizes RNP complexes close to the axes of the active Y chromatin. In the "pseudonucleolus" it can be shown that the structures recognized by pp60 are quite distinct from those detected by Dm 28K2. Thus, the "pseudonucleolus" is a striking example for the presence of different RNP populations within a same defined nuclear compartment. Together with previous results (Glatzer, K. H., 1984, Mol. Gen. Genet., 196:236- 243), our data represent evidence that the morphological and apparently functional differences between the active Y chromosomal loops, which are involved in male fertility, are caused by the presence of qualitatively and possibly also functionally different RNP populations within these nuclear compartments. Because both RNP antigens are discussed in the literature in connection with repressed mRNP the observed cross-reaction of the respective antibodies in D. hydei suggests a more general and important function of these proteins in the RNA metabolism of eukaryotic cells.     

Fluram induces species-dependent C and G bands in mammalian chromosomes, revealing heterogeneous distribution of chromosomal proteins

Fluram (Fluorescamine; 4-phenylspiro(furan-2(3H),1'-phthalan)-3,3'-dione) is a fluorogenic reagent, which permits the detection of primary amines by forming highly fluorescent pyrrolinone derivatives. This reagent has been used on methanol-acetic acid fixed metaphase chromosomes of mouse and man and proved to be very effective in differentiating chromosome regions in both genomes. Mouse centromeric heterochromatin is highly reactive, showing intense fluorescence in all centromeric regions, whereas human chromosomes show no fluorescence in such regions. In addition, a G-like banding pattern is also obtained in both types of chromosomes. The differential reactivity of each chromosome region showed by this method demonstrates a heterogeneous distribution of chromosome proteins, resulting in a chromosome banding pattern, which is in this case species dependent.