Hypomethylation of human sperm pronuclear chromosomes

Using the methylase SssI enzyme, we have analyzed the degree of in situ methylation of human sperm pronuclear chromosomes obtained by fertilizing hamster oocytes with human sperm. Untreated (control) sperm chromosome complements showed a higher degree of in situ methylation, compared to sperm complements previously treated with 5-azadeoxycytidine or lymphocyte chromosomes. This indicates that human sperm pronuclear chromosomes have a lower degree of genomic methylation compared to that of other somatic cells. The similarity in the degree of in situ methylation of the euchromatic and heterochromatic regions of chromosomes 1, 9, 15, and 16 and the Y chromosome in human sperm does not support the existence of a possible correlation between hypomethylation and heterochromatin decondensation.     

Centromeres, checkpoints and chromatid cohesion

An emerging view is that the formation of active centromeres is modulated in an epigenetic manner reflecting the association of centromeres with heterochromatin. Support for this comes from studies on fission yeast centromeres, the properties of human neocentromeres and dicentric chromosomes, and analyses of Drosophila minichromosome deletion derivatives. A link has been established between tension across kinetochores and the phosphorylation status of kinetochore components. Vertebrate homologues of yeast MAD2 have recently been isolated and localized to kinetochores, indicating that components of the spindle integrity checkpoint are conserved. The linkage between sister chromatids is only dissolved at anaphase during mitotic and meiotic divisions. Phenotypic and localization data combined with their pattern of rapid degradation at anaphase have implicated several yeast and Drosophila proteins in aspects of sister chromatid cohesion.     

Alphoid repetitive DNA in human chromosomes

The thesis describes the first extensive DNA sequence analysis that demonstrated that the tandemly repeated alphoid DNA in the centromere of the human chromosomes consists of distinct subfamilies and in a number equal to or exceeding the number of chromosomes. The expected presence of only one or a few distinct subfamily on individual chromosomes was supported by the characterization of an extremely well-defined subfamily specific for chromosome 7 and represented in the original collection of subfamilies. The pattern of chromosome-specificity breaks down among the acrocentric chromosomes where chromosomes 13 and 21 were found to share one and chromosomes 14 and 22 to share another specific subfamily. By in situ hybridization these subfamilies were shown not to be shared by other chromosomes. The remarkable pairwise pattern of sequence homogenization was present also in the chimpanzee genome raising the question of its biological role. However, the subfamilies on these human and chimpanzee chromosomes are not orthologous but were shown to originate from two evolutionarily different repeat families. It follows that dramatic sequence evolution has occurred in one or both species during or after separation. The sequence evolution might even occur at a higher rate in humans. This possibility was studied in orthologous alphoid sequences on the X chromosome of humans and the great apes. The analysis supports the general view that our closest relative is the chimpanzee and indicates that the rate of recombination is increased in the human repeat DNA. A "molecular clock" running faster in this DNA may have evolutionary implications. Finally, the usefulness of alphoid subfamilies as chromosome-specific markers is illustrated in a cytogenetic dissection of the centromeric region of Robertsonian translocations. The breakpoints were located to satellite III DNA leaving these chromosomes dicentric. The order of the different tandem DNAs on the p-arm of the acrocentric chromosomes could also be established.     

Visual classification of banded human chromosomes. I. Karyotyping compared with classification of isolated chromosomes

Visual karyotyping and visual classification of isolated chromosomes was carried out by seven investigators on 22 trypsin banded metaphases of average quality. The karyotyping experiment resulted in an average error rate of 0-1% (zero-0-4%) and the classification of isolated chromosomes resulted in an error rate of 3% (2-5%). The B and F group chromosomes were found to be most difficult to classify when isolated, while no errors were made of the no. 1 and the X chromosome. Large differences were seen in the resulting error pattern for the individual investigators both with regard to their total error rate and also the chromosome types which they most frequently misclassified. Based upon these error patterns it is suggested that more than 95% of the chromosomes in an average quality material contain features upon which a reliable visual classification can be made. Thus there may be a potential possibility that these chromosomes may be classified by computer on the basis of these features. The fact that visual karyotyping is much more reliable than visual classification of isolated chromosomes indicated that computer classification of chromosomes should include programming capable of making appropriate comparison between the chromosomes in the metaphase and at the same time take into account the expected presence of 23 chromosome pairs for normal cells. This would simulate the human performance of visual karyotyping and make a classification possible of at least some of the remaining 5% difficult chromosomes.     

Segregational fidelity of chromosomes in human thyroid tumour cells

Using fluorescence in situ hybridisation (FISH) we have analysed the segregational fidelity of all the human chromosomes during mitotic cell division. The losses and gains of chromosomes were analysed in human polyploid cell lines derived from a well-differentiated papillary thyroid cancer. These thyroid cells can be cultured for more than 300 population doublings. For the purpose of our study the polyploid nature of the cells may act as a protective buffer against the cell-lethal effects of the loss of individual chromosomes. To evaluate the role of the p53 gene product in maintaining the fidelity of chromosome segregation we compared the frequencies of chromosome loss and gain in cultures with wild-type p53 activity (K1E7neo3) and cultures transfected with plasmids expressing a mutant p53 product (K1E7scx6). Cultures were analysed for the presence of both structurally normal and rearranged chromosomes at both early and late passages. Cell cultures with defective p53 activity showed progressive chromosome loss from a median chromosome number of 87-97 to 75-86. Cell growth in cultures with wild-type p53 activity showed the loss of chromosomes 6, 7, and 8 and the gain of 17 and 20. Cultures expressing mutant p53 activity showed the loss of chromosomes 2, 5, 14 and 17 and the gain of 4 and 22. The combination of defective p53 and growth resulted in further destabilisation with the additional losses of chromosomes 3, 11, 15, 16 and 21. Chromosomes 1, 9, 10, 12, 13, 18, 19, X and Y segregated stably under all the culture conditions as did the structurally rearranged marker chromosomes. The study has demonstrated variation in the fidelity of mitotic chromosome segregation and the influence of p53 gene activity upon the segregation of individual human chromosomes.     

The role of chromosomes in the aetiology of human abortion

Of 27 specimens of abortus tissue from first-trimester spontaneous abortions investigated, 26% had chromosome abnormalities. In contrast, the investigation showed that the incidence of chromosome abnormalities in 36 midtrimester cases was only 2,8%. Two of 19 ectopic pregnancies had abnormal chromosome complements.     

Volume determination of human metaphase chromosomes by scanning force microscopy

The scanning force microscopy (SFM) yields the topography of the investigated surface. A procedure was developed which starts from this three-dimensional information to estimate the volume of a biological specimen. The volume of spread human metaphase chromosomes was determined in air and rehydrated in aqueous buffer. A difference of the determined volume of a air-dried metaphase chromosome set was found compared to values from electron microscopic investigations, and could be correlated with differences in the hydration state of the chromosomes. SFM-based relative volumes of air-dried chromosomes resembles literature data regarding volume range and distribution. Possible application of SFM-based relative volume measurements for chromosome classification purposes is discussed.     

Inheritance of acridine orange R variants in human acrocentric chromosomes

A case is reviewed of bilateral temporal bone metastases from a carcinoma of the uterine cervix. The pertinent literature is reviewed and the temporal bone histology is described.     

Two-parameter flow fluorescence analysis of human chromosomes. Quantitative processing]

Adrenomedullin (AM) is a newly discovered hypotensive peptide which is believed to play an important role for blood pressure control in the adult. Although it has been well established that a major production site of AM is vascular endothelial cells, we now show that AM is most highly expressed in trophoblast giant cells, which are derived from the conceptus and are directly in contact with maternal tissues at the implantation site. Northern blot and in situ hybridization analyses show that the AM mRNA begins to be detected just after implantation and its level peaks at 9.5 days postconception (d.p.c.) in those cells. Expression then falls dramatically after 10.5 d.p.c., coincident with the completion of the mature chorioallantoic placenta. Immunohistochemical analyses show that the AM peptide is secreted from the trophoblast giant cells into the surrounding tissues, i.e., embryo, decidua, and maternal circulation. In contrast, the expression of an AM receptor was not detected by Northern blot analyses in either embryo or trophoblast giant cells at 7 d.p.c., when the AM gene is most highly expressed in the trophoblast giant cells. This suggests that the AM produced and secreted from the embryo's trophoblast giant cells acts on the maternal tissues rather than on the embryonic tissues. Based on these results, we propose that the high production of AM may be the mechanism by which the embryos survive at the early postimplantation period by pooling maternal blood in the implantation site in order to secure nutrition and oxygen before the establishment of efficient embryo-maternal circulation through the mature placenta.     

Ribosomal DNA clusters in pulsed-field gel electrophoretic analysis of human acrocentric chromosomes

For determination of the extent to which ribosomal DNA (rDNA) is organized in tandemly repeated arrays, cellular DNA was digested with a restriction enzyme (EcoRV) that does not cut within the single 44-kb rDNA unit, and fragments separated by PFGE were hybridized to specific rDNA probes. A series of bands large enough to contain 15 to more than 30 rDNA repeat units was observed. In YACs containing cloned rDNA, however, such clusters were not observed, presumably because, as shown here for a clone starting with 1.5 tandem repeat units, there is a tendency for repeat units to delete out of the insert. By comparative gel electrophoretic analyses of DNAs from rodent hybrid cells containing singly isolated human chromosomes, most of the bands seen in total human DNA were assigned to at least one of the acrocentric chromosomes. Thus, large characteristic assemblies of DNA containing rDNA and lacking EcoRV sites were stable enough to be conserved in some human/rodent hybrid lines. When further digested with HindIII, which cuts rDNA at several points, the rDNA in each band yielded the expected fragments. If the large species consist completely of clusters of tandemly repeated rDNA units, they account for about half of the total cellular rDNA content estimated by saturation hybridization measurements.     

Effects of bis(2-ethylhexyl) phthalate on chromosomes of human leukocytes and human fetal lung cells

Blood from two male and two female donors was exposed at 37degrees for 4 hr to concentrations of 60.0, 6.0, 0.6, and 0.06 mug of a widely used plasticizer, bis (2-ethylhexyl) phthalate, per milliliter of blood. The bis(2-ethylhexyl) phthalate was solubilized with polysorbate 80. Appropriate polysorbate and nonpolysorbate controls also were established. Following the 4 hr of incubation, phytohemagglutinin was added and tissue cultures were established. In addition, human fetal lung cells were exposed in tissue culture to a medium containing 6.0 mug/ml of bis(2-ethylhexyl) phthalate in polysorbate 80 for 5 days. Similar controls also were established for these experiments. Analysis of chromosome preparations from all cultures obtained failed to show any increased evidence of isochromatid and chromatid breaks or gaps or abnormal forms at any studied concentration when compared to the control cultures. In addition, analysis of fetal lung cell preparations for aneuploidy failed to reveal any differences between cells from study and control cultures. This study involved a short-term exposure to bis(2-ethylhexyl) phthalate in various concentrations which did not cause damage in leukocytes or fetal lung cells.     

A new region of conservation is defined between human and mouse X chromosomes

Comparative mapping of the X chromosome in eutherian mammals has revealed distinct regions of conservation as well as evolutionary rearrangements between human and mouse. Recently, we and others mapped the murine homologue of CLCN4 (Chloride channel 4) to band F4 of the X chromosome in Mus spretus but to chromosome 7 in laboratory strains. We now report the mapping of the murine homologues of APXL (Apical protein Xenopus laevis-like) and OA1 (Ocular albinism type I), two genes that are located on the human X chromosome at band p22. 3 and in close proximity to CLCN4. Interestingly, Oa1 and Apxl map to bands F2-F3 in both M. spretus and the laboratory strain C57BL/6J, defining a new rearrangement between human and mouse X chromosomes.     

Mapping of eight human chromosome 1 orthologs to cattle chromosomes 3 and 16

PTFE-e patches were used in nasal augmentation in 14 patients with a four-year follow-up. This material was chosen because it is inert and biologically compatible with human tissue. None of the patients in which this material was used had intolerance reaction, skin changes, material extrusion, or signs of infection. A review of the world literature showed no cases of poor results with PTFE-e. It is concluded that the best material for nasal augmentation is autologous material, but if not available or difficult to obtain, PTFE-e is the most acceptable synthetic material and produces good results.     

Cytogenetic effects of cryopreservation on human sperm: assessment using an improved method for analyzing human sperm chromosomes

OBJECTIVE: To evaluate any cytogenetic effects of cryopreservation on human sperm by comparing the frequencies of sperm chromosome anomalies and sex ratios before and after freezing. METHODS: Using in vitro fertilization of zona-free hamster oocytes, analysis of sperm chromosomes was first performed on portions of fresh human semen samples. The residual semen was then analyzed for sperm chromosomes after cryopreservation for several weeks. Sperm donors were 5 healthy men aged 26-38 years. RESULTS: A total of 166 sperm karyotypes were analyzed, 94 before freezing and 72 after freezing. The results indicated no significant differences between fresh and frozen sperm in either frequencies of aneuploidy (fresh: 0%, frozen: 2.8%) or structural anomalies (fresh: 7.5%, frozen: 9.7%). The sex ratios did not differ from the expected 1:1 ratio under either condition. CONCLUSIONS: The results of these studies indicate that cryopreservation does not exert any cytogenetic mutagenicity on human spermatozoa or alter X/Y ratio of human sperm.     

Suppression of tumorigenicity and induction of senescence on human endometrial carcinoma cell lines by transfer of normal human chromosomes

The author examined the ability of human chromosomes derived from normal fibroblast cells to suppress the tumorigenicity of HHUA and Ishikawa cells, human endometrial carcinoma cell lines. Using DNA transfection, the human chromosome tagged with a selectable marker (the pSV2neo gene, which encodes resistance to the antibiotic, G418) was transferred to mouse A9 cells by cell hybridization and microcell fusion techniques. Thus, a library of mouse A9 clones containing individually a different human chromosome tagged with the pSV2neo plasmid DNA was constructed. Transfer by microcell fusion of either chromosome 1, 6, 9, 11 or 19 into the HHUA and Ishikawa cell lines was performed, and the abilities of the microcell hybrids to form tumors in nude mice were examined. The introduction of chromosome 19 had no effect on the tumorigenicity, whereas microcell hybrid clones with an introduced chromosome 1, 6 and 9 completely suppressed the tumorigenicity of the both lines. A decrease in tumor-take incidence in some but not all clones of HHUA cells was observed following the introduction of a chromosome 11. The nontumorigenic microcell hybrids with an introduced chromosome 1 differed from the nontumorigenic microcell hybrids with an introduced chromosome 6, 9, or 11. A large percentage of hybrids with chromosome 1 sensed and/or showed alterations in cellular morphology and transformed growth properties in vitro on the both cell lines. These results indicate that more than one chromosome carries a tumor suppressor gene(s) for human endometrial carcinoma cell lines, and indicate that normal human chromosome 1 carries gene(s) which suppresses the immortalization. This supports the hypothesis that multiple tumor suppressor gene(s) control the various tumorigenic phenotypes at the different step during process of neoplastic development.     

Epstein-Barr-based episomal chromosomes shuttle 100 kb of self-replicating circular human DNA in mouse cells

We describe the microcell fusion transfer of 100-200 kb self-replicating circular human minichromosomes from human into mouse cells. This experimental approach is illustrated through the shutting of the latent 170 kb double-stranded DNA genome from the human herpesvirus, Epstein-Barr virus, into nonpermissive rodent cells. Using this interspecies transfer strategy, circular episomes carrying 95-105 kb of human DNA were successfully established at low copy number in mouse A9 cells. Selected episomes were stably maintained for 6 months, and unselected episomes were characterized by a 95% episomal retention per cell division. The establishment of a mouse artificial episomal chromosome system should facilitate evolutionary and therapeutic studies of large human DNA in rodent genetic backgrounds.     

Characteristics of chromosomes in polarized normal human bronchial cells provide a blueprint for nuclear organization

In normal human terminally differentiated and polarized bronchial cells, the fluorescent "painting" technique (FISH) for all chromosomes (except Y) documented that each homologue of each chromosome occupies a distinct, separate domain within the nucleus. The homologues are distributed along the nuclear membrane. In most cells and chromosomes studied, the two homologues were not identical: one was usually more "compact" than the other which was more "open," displaying fiber-shaped extensions. The differences between the territories of homologues 1 and 7 were shown to be statistically valid (P < 0.0001 by Wilcoxon sign rank test), as has been previously documented for the two X chromosomes (Eils et al., 1996). In some parallel arrays of bronchial cells, the position of the chromosomes in the nuclei was either identical or formed a mirror image, suggesting that the position of the chromosomes in polarized nuclei may be constant. To confirm this observation, the angles formed by the two homologues in the polarized oval nuclei were measured for chromosomes 1, X, and 7. The measurements disclosed that, in about two-thirds of the nuclei, the two homologues formed angles of 150 degrees, 157 degrees and 148 degrees, nearly identical to those formed by the same three chromosomes in prometaphase rosettes of cultured diploid human fibroblasts (Nagele et al., 1995). In about one third of the nuclei, the same homologues formed angles of 89 degrees, 72 degrees, and 94 degrees, and occasionally an angle of 180 degrees. A three-dimensional computer reconstruction of the nuclei was performed using the data for the X chromosomes. By cinematographic technique, it could be documented that the angles separating the two homologues depended on the rotation of the nucleus along the axes X, Y, and Z. The cause of the rotation is speculative at this time. Because of the concordance of these data in terminally differentiated epithelial cells with prior observations on prometaphases of human diploid fibroblasts, it is suggested that the position of chromosomes in all human cells is constant throughout the cell cycle. The possible significance of these observations is discussed.