Evidence that the human X chromosome is enriched for male-specific but not female-specific genes

There is increasing evidence that X chromosomes have an unusual complement of genes, especially genes that have sex-specific expression. However, whereas in worm and fly the X chromosome has a dearth of male-specific genes, in mice genes that are uniquely expressed in spermatogonia are especially abundant on the X chromosome. Is this latter enrichment true for nongermline, male-specific genes in mammals, and is it found also for female-specific genes? Here, using SAGE data, we show (1) that tissue-specific genes tend to be more abundant on the human X chromosome, (2) that, controlling for this effect, genes expressed exclusively in prostate are enriched on the human X chromosome, and (3) that genes expressed exclusively in mammary gland and ovary are not so enriched. This we propose is consistent with Rice's model of the evolution of sexually antagonistic alleles.     

Transfer of the human X chromosome to human--Chinese hamster cell hybrids via isolated HeLa metaphase chromosomes.

Evidence is presented for the uptake of the human X chromosome by human-Chinese hamster cell hybrids which lack H P R T activity, following incubation with isolated human HeLa S3 chromosomes. Sixteen independent clonal cell lines were isolated in H A T medium, all of which contained a human X chromosome as determined by trypsin-Giemsa staining. The frequency of H A T-resistant clones was 32 x 10(-6) when 10(7) cells were incubated with 10(8) HeLa chromosomes. Potential reversion of the hybrid cells in H A T medium was less than 5 x 10(-7). The 16 isolated cell lines all contained activity of the human X-linked marker enzymes H P R T, P G K,alpha-Gal A, and G6PD, as determined by electrophoresis. The phenotype of G6PD was G6PD A, corresponding to G6PD A in HeLa cells. The human parental cells used in the fusion to form the hybrids had the G6PD B phenotype. The recipient cells gave no evidence of containing human X chromosomes. These results indicate that incorporation and expression of HeLa X chromosomes is accomplished in human-Chinese hamster hybrids which lack a human X chromosome.     

A practical metaphase marker of the inactive X chromosome.

It is paradoxical that the inactivated X is the only chromosome that can be identified in the interphase nucleus, yet in metaphase, it is indistinguishable from its genetically active homolog unless special culture and staining procedures are employed. A specific inactivation-associated fold in proximal Xq resolves that paradox. We describe here how the fold in the proximal long arm can be used as a simple and reliable marker to identify the inactivated X in G-, Q-, or R-banded preparations. Several examples are given, including localization of the inactivation center to band Xq13 or q21.1, identification of nonrandom inactivation in X-chromosome rearrangements, identification of multiple active X chromosomes in tumor cell lines, analysis of X-inactivation patterns in female carriers of the fragile site at Xq27, and comparison of X-inactivation patterns among primate species.     

The human inactivated X chromosome folds in early metaphase,prometaphase, and prophase

Summary The inactivated X chromosome has a site of unusually frequent folding in region Xq1, whereas a fold in Xq1 is uncommon on the active X. We investigated the pattern of X chromosome folding in high-resolution GTG- and RBG-stained preparations from four women. In early metaphase cells, slightly more than 50% of late-replicating Xs folded at Xq1Xq21, compared with about 6% of early replicating Xs. The late-replicating X folded in about 80% of prometaphase cells; the early, in only about 14% of these cells. And the latereplicating X folded in 19 of 20 prophase cells. Occasionally, one X had an omega-shaped loop or apparent physical connection between Xq13 and Xq21.1. It is possible that a segment of Xq1 never completely uncoils and may help to provide continuity for the Barr body from one interphase to the next.     

The obscure parts of the X chromosome inDrosophila melanogaster

Through the use of small atelic fragments derived from T (1; 4) A,B ^S the right arm of the X chromosome ofD. melanogaster has been demonstrated.The proximal portion of XR is a non-chromatic or ghost reversed repeat of the proximal portion of XL.Polytene X chromosomes show no indications of the limits of the heterochromatic blocks which characterize mitotic X chromosomes.The nuclear organizer is a subterminal portion of the polytene XR, whereas it is situated in XL in mitotic neuroblast cells.It is suggested that the non-chromatic XR may contain a ghost allele of the genebobbed, and of perhaps other adjacent genes.This investigation was supported in part by Contract No. AT (30-1)-2113 from the U.S. Atomic Energy Commission, and in part by Public Health Service Research Grant No. C-4362 from the National Institutes of Health.     

Construction and analysis of linking libraries from the mouse X chromosome

A hybrid cell line containing the mouse X chromosome on a human background has been used to construct linking libraries from the mouse X chromosome, and approximately 250 unique EagI and NotI clones have been identified. Seventy-three clones have been sublocalized onto the X chromosome using interspecific Mus spretus/Mus domesticus crosses and a panel of somatic cell hybrids carrying one-half of reciprocal X-autosome translocations. The average spacing of the linking clones mapped to date is about one every 2 Mb of DNA. Two clones from the central region of the chromosome have been physically linked by pulsed-field gel electrophoresis. A large number of clones contain conserved sequences, indicating the presence of CpG-rich island-associated genes. The clones isolated from these libraries provide a valuable resource for comparative mapping between man and mouse X chromosomes, isolation of X-linked disease loci of interest by reverse genetics, and analysis of the long-range structure and organization of the chromosome.     

Characterization of four human YAC libraries for clone size, chimerism and X chromosome sequence representation.

Four collections of human X-specific YACs, derived from human cells containing supernumerary X chromosomes or from somatic cell hybrids containing only X human DNA were characterized. In each collection, 80-85% of YAC strains contained a single X YAC. Five thousand YACs from the various libraries were sized, and cocloning was assessed in subsets by the fraction of YAC insert-ends with non-X sequences. Cocloning was substantial, ranging up to 50% for different collections; and in agreement with previous indications, in all libraries the larger the YACs, the higher the level of cocloning. In libraries made from human-hamster hybrid cells, expected numbers of clones were recovered by STS-based screening; but unexpectedly, the two collections from cells with 4 or 5 X chromosomes yielded numbers of YACs corresponding to an apparent content of only about two X equivalents. Thus it is possible that the DNA of inactive X chromosomes is poorly cloned into YACs, speculatively perhaps because of its specialized chromatin structure.     

Construction of gene libraries for each human chromosome

We describe the construction of two complete sets of small insert, complete digest DNA libraries for each of the 24 human chromosomal types by the National Laboratory Gene Library Project. Flow sorting was used to purify the chromosomes which provided the DNA for cloning. One set of libraries was cloned into the HindIII site of the lambda vector Charon 21A, and the other set was cloned into the EcoRI site of the same vector. Characterization information from both in-house experiments and user feedback is presented. These chromosome-specific libraries are available to the general scientific community from a repository at the American Type Culture Collection, Rockville, MD. The second phase of the project, the construction of large insert, partial digest libraries in both lambda and cosmid vectors, is underway.     

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

Summary 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.     

A genome approach to the human X chromosome.

The human X chromosome includes many disease genes, some of which have already been cloned using time-consuming and labor-intensive methods. A more efficient way to study this chromosome makes use of technology emerging from the human genome initiative.     

Construction and use of Plasmodium falciparum phage display libraries to identify host parasite interactions

BACKGROUND: The development of Plasmodium falciparum within human erythrocytes induces a wide array of changes in the ultrastructure, function and antigenic properties of the host cell. Numerous proteins encoded by the parasite have been shown to interact with the erythrocyte membrane. The identification of new interactions between human erythrocyte and P. falciparum proteins has formed a key area of malaria research. To circumvent the difficulties provided by conventional protein techniques, a novel application of the phage display technology was utilised. METHODS: P. falciparum phage display libraries were created and biopanned against purified erythrocyte membrane proteins. The identification of interacting and in-frame amino acid sequences was achieved by sequencing parasite cDNA inserts and performing bioinformatic analyses in the PlasmoDB database. RESULTS: Following four rounds of biopanning, sequencing and bioinformatic investigations, seven P. falciparum proteins with significant binding specificity toward human erythrocyte spectrin and protein 4.1 were identified. The specificity of these P. falciparum proteins were demonstrated by the marked enrichment of the respective in-frame binding sequences from a fourth round phage display library. CONCLUSION: The construction and biopanning of P. falciparum phage display expression libraries provide a novel approach for the identification of new interactions between the parasite and the erythrocyte membrane.     

Construction and use of Plasmodium falciparum phage display libraries to identify host parasite interactions

The development of an electroporation based transfection method for Plasmodium falciparum has been very successful for the study of some genes but its efficiency remains very low. While alternative approaches have been documented, electroporation of infected red blood cells generally remains the method of choice for introducing DNA into P. falciparum. In this paper we compare four published transfection techniques in their ability to achieve stable transfections.     

Packing of the 30 nm chromatin fiber in the human metaphase chromosome

The human genetic material is packed hierarchically within the metaphase chromosome: the DNA moleculet together with histone proteins form 11 nm diameter nucleosomes, which are then ordered into the 30 nm thick chromatin fiber. Little is known about the packing of this fiber within the chromosome. We have developed a tracking algorithm with which we followed its path within a three-dimensional reconstruction of a human chromosome computed from a series of electron micrographie projections. Fiber segments were seen to form loops of 100–350 nm diameter. Our observations indicate that these loops — which themselves show no preferred orientation — are organised into regions of roughly 200 nm axial extent.     

X chromosome constitution and the human female phenotype

Summary The correlations of abnormal X chromosome constitutions and the resulting phenotypes in the human female are reviewed. The following hypotheses put forward to explain these correlations are discussed in detail: (1) The damage is done before X inactivation; (2) An effect is exerted between reactivation of the X chromosome(s) and meiosis in oocytes; (3) A recessive gene(s) in hemizygous condition might be expressed in the cases in which the same X is active in all cells; (4) A change in the number of presumed active regions on the inactive X chromosomes might have an effect; (5) A position effect, in that the region Xq13-q27 has to be intact in both X chromosomes to allow normal development, may be responsible; (6) An effect during the period when cells with different inactivation patterns compete is a probability; (7) The original X inactivation may be neither regular nor random.The conclusion reached is that the phenotypic effects of a specific X chromosome aberration may be simultaneously exerted through different pathways (Tables 1 and 2). Hypotheses (2), (4), (5), and (6) are considered probable. Hypothesis (3) has been discarded, and there is very little evidence for hypotheses (1) and (7).     

Duplications of the X chromosome in males: evidence that most parts of the X chromosome can be active in two copies

Summary We have analysed two duplications of the X chromosome in male patients using chromosome replication and DNA methylation patterns as determinants of the functional status of the duplicated segments. In both cases, the large duplicated regions, Xq12-q22 and Xq26.3-qter, were not inactivated. A review of previously reported male cases revealed that these duplications were also not subject to inactivation. Taken together, the examined duplications cover almost the entire X chromosome except the pericentromeric region and Xq25–26. Thus, most regions of the X chromosome can be present in two functional copies without lethal consequences.     

Immunocytochemical study of the action of cis-dichlorodiamino platinum on the human metaphase chromosome

The reaction of cis-dichlorodiamino platinum (cis-Pt) with the human metaphase chromosome was studied using both Giemsa staining and the action of anti-cis-Pt antibody. Two kinds of experiment were performed: (1) cis-Pt was placed in contact with cells in culture (in vivo tests), (2) cis-Pt was placed in contact with chromosomes fixed on slides (in vitro tests). In vivo, the presence of cis-Pt during phase G1 of the cell cycle is important for the reaction; in vitro, competition between cis-Pt and Giemsa for fixation on chromosomal DNA was shown to occur.     

Evidence for archaic Asian ancestry on the human X chromosome

The human RRM2P4 pseudogene has a pattern of nucleotide polymorphism that is unlike any locus published to date. A gene tree constructed from a 2.4-kb fragment of the RRM2P4 locus sequenced in a sample of 41 worldwide humans clearly roots in East Asia and has a most-recent common ancestor approximately 2 Myr before present. The presence of this basal lineage exclusively in Asia results in higher nucleotide diversity among non-Africans than among Africans. A global survey of a single-nucleotide polymorphism that is diagnostic for the basal, Asian lineage in 570 individuals shows that it occurs at frequencies up to 53% in south China, whereas only one of 177 surveyed Africans carries this archaic lineage. We suggest that this ancient lineage is a remnant of introgressive hybridization between expanding anatomically modern humans emerging from Africa and archaic populations in Eurasia.