Study of human chromosome reproduction time using 5-bromdeoxyuridine]

5-Bromodeoxyuridine (BDU) -- 20 gamma/ml, 5-fluorodeoxyuridine (FDU) -- 0.1 gamma/ml and uridine -- 3 gamma/ml were introduced into the lymphocyte cultures of the peripheral blood of normal persons of both sexes. Chromosome preparations were prepared in 5 hours they were stained after the incubation in the fluorochrome solution Hoechstt 33258 with a 2% Giemsa solution on phosphate buffer (pH -- 5.0). Chromosome areas with incorporated BDU stained with Giensa solution but weakly; because of this regions with late replication were seen in each chromosome in thr form of weakly stained segments. A picture of late DNA replication is described on the basis of this method in all human chromosomes.     

Localization of the gene encoding human Factor V to chromosome 1q21–25

The gene encoding human coagulation Factor V (FV), one of the cofactors in the blood clotting process, has been mapped to chromosome 1 by both Southern hybridization to DNA from human-hamster somatic cell hybrids and in situ hybridization. The whole plasmid pUC3A containing a 1.5-kb cDNA sequence for FV was 32P-labeled for Southern analysis and 3H-labeled for in situ hybridization to metaphase chromosomes. The results localized the FV gene to the region of 1q21-25.     

Transposition of human immunoglobulin V kappa genes within the same chromosome and the mechanism of their amplification.

The variable, joining and constant gene segments of the human immunoglobulin kappa locus (V kappa, J kappa and C kappa) are located on the short arm of chromosome 2 at 2p11-2p12. Here we describe a cluster of 11 V kappa genes on the long arm of chromosome 2 at 2cen-q11. By pulsed-field gel electrophoresis, cosmid cloning and DNA sequencing the cluster was shown to consist of four amplified units (amplicons). The amplicons, each 110-160 kb in size, are organized within 650 kb as an array of inverted repeats with short stretches of non-amplified DNA in between. Cloning and sequencing of three different joints between amplified and non-amplified DNA revealed the existence of parts of Alu repeats at each of the analysed joints. It is suggested that during evolution a group of five V kappa genes was transposed from the short to the long arm of chromosome 2 by a pericentric inversion. Three of the five V kappa genes were then amplified in two subsequent steps to yield the structure found in the majority of the present day population. The possible relation of this structure to a pericentric inversion of chromosome 2 that is seen cytogenetically in a small fraction of today's population is discussed.     

Mechanisms of chromosome banding. V. Quinacrine banding.

A series of biochemical investigations were undertaken to determine the mechanism of Q-banding. The results were as follows: 1. In agreement with previous studies, highly AT-rich DNA, such as poly(dA)-poly(dT), markedly enhanced quinacrine fluorescence while GC containing DNA quenched fluorescence. These effects persisted at DNA concentrations comparable to those in the metaphase chromosome. 2. Studies of quinacrine-DNA complexes in regard to the hypochromism of quanacrine, DNA Tm, DNA viscosity, and equilibrium dialysis, indicated the quinacrine was bound be intercalation with relatively little sid binding. 3. Single or double stranded nucleotide polymers, in the form of complete or partial helices, were 1000-fold more effective in quenching than solutions of single nucleotides, suggesting that base stacking is required for quenching. 4. Studies of polymers in the A conformation, such as transfer RNA and DNA-RNA hybrids, indicated that marked base tilting does not affect the ability of nuclei acids to cause quenching or enhancement of quinacrine fluorescence. 5. Salts inhibit the binding of quinacrine to DNA. 6. Spermine, polylysine and polyarginine, which bind in the small groove of DNA, inhibited quinacrine binding and quenching, while histones, which probably bind in the large groove, had little effect. This correlated with the observation that removal of histones with acid has no effect on Q-banding. 7. Mouse liver chromatin was separated into five fractions. At concentrations of quinacrine from 2 times 10-6 to 2 times 10-5 M all fractions inhibited to varying degrees the ability of the chromatin DNA to bind quinacrine and quench quinacrine fluorescence. At saturating levels of quinacrine two fractions, the 400 g pellet (rich in heterochromatin) and a dispersed euchromatin supernatant fraction, showed a decreased number of binding sites for quinacrine. These two fractions were also the richest in non-histone proteins. 8. DNA isolated from the different fractions all showed identical quenching of quinacrine fluorescenc. 9. Mouse GC-rich, mid-band, AT-rich, and satellite DNA, isolated by CsCL AND Cs-2SO-4-Ag+ centrifugation all showed identical quenching of quinacrine fluorescence, indicating that within a given organism, except for very AT or GC-rich satellites, the variation in base composition is not adequate to explain Q-banding.We interpret these results to indicate that: (a) quinacrine binds to chromatin by intercalation of the three planar rings with the large group at position 9 lying in the small groove of DNA, (b) most pale staining regions are due to a decrease binding of quinacrine, and (c) this inhibition of binding is predominately due to non-histone proteins.     

A comparative map of chicken chromosome 24 and human chromosome 11

To improve the physical and comparative map of chicken chromosome 24 (GGA24; former linkage group E49C20W21) bacterial artificial chromosome (BAC) contigs were constructed around loci previously mapped on this chromosome by linkage analysis. The BAC clones were used for both sample sequencing and BAC end sequencing. Sequence tagged site (STS) markers derived from the BAC end sequences were used for chromosome walking. In total 191 BAC clones were isolated, covering almost 30% of GGA24, and 76 STS were developed (65 STS derived from BAC end sequences and 11 STS derived within genes). The partial sequences of the chicken BAC clones were compared with sequences present in the EMBL/GenBank databases, and revealed matches to 19 genes, expressed sequence tags (ESTs) and genomic clones located on human chromosome 11q22-q24 and mouse chromosome 9. Furthermore, 11 chicken orthologues of human genes located on HSA11q22-q24 were directly mapped within BAC contigs of GGA24. These results provide a better alignment of GGA24 with the corresponding regions in human and mouse and identify several intrachromosomal rearrangements between chicken and mammals.     

Senescence and human chromosome changes

Summary A girl with slight psychomotor retardation, microphthalmia, and colobomata of the left eye, a hypotrophy of the right arm and a surnumerary digit on the right hand is described. The routine chromsome analysis and a G-banding analysis revealed an elongated long arm of chromosome 10. An extra light and dark band was present proximally. Both parents had normal chromosomes. While the visual comparison of the abnormal with the normal chromosome 10, did not enable the extra bands of the normal bands q21 and q22 to be distinguished. However, measurements of length, surface area, and relative reflection of the different light and dark bands of the long arm on tracings or directly on the normal and abnormal chromosomes, enabled us to precisely locate the extra bands and to determine that the abnormal chromosome was a result of an insertional translocation. The value of such measurements is discussed.     

Interphase chromosome structures of human cells

Major intermediates of chromosome condensation in erythroleukemia K562 cells are presented. Interphase chromatin structures became visible after reversal of permeabilization. Large-scale chromatin structures and the development of individual interphase chromosomes were observed by fluorescence microscopy. In the linear arrangement the following major intermediates of K562 chromatin condensation could be distinguished: (1) the most decondensed chromatin veil, (2) chromatin ribbon, (3) chromatin funnel, a new intermediate regarded as the earliest visible form of interphase chromosomes, (4) chromatin body, (5) 300 nm chromatin fiber, (6) u, v, or s forms of chromosomes, and (7) linear chromosomes. The observations made in nuclei of K562 cells conform to the model of helical coil chromosome condensation.     

Characterization of a human chromosome 22 enriched bacterial artificial chromosome sublibrary

Selection of chromosomal sublibraries from total human genomic libraries is critical for chromosome-based physical mapping approaches. We have previously reported a method of screening total human genomic library using flow sorted chromosomal DNA as a hybridization probe and selection of a human chromosome 22-enriched sublibrary from a total human bacterial artificial chromosome (BAC) library (Nucleic Acids Res 1995; 23: 1838–1839). We describe here further details of the method of construction as well as characterization of the chromosome 22-enriched sublibrary thus constructed. Nearly 40% of the BAC clones that have been mapped by fluorescence in situ hybridization (FISH) analysis were localized to chromosome 22. By screening the sublibrary using chromosome 22-specific hybridization probes, we estimated that the sublibrary represents at least 2.5 × coverage of chromosome 22. This is in good agreement with the results from FISH mapping experiments. FISH map data also indicate that chromosome 22-specific BACs in the sublibrary represent all the subregions of chromosome 22.     

Ultrastructure of the human chromosome fiber

Summary Human chromosomes and their fibers were studied by electron microscopy as whole mounts in metaphase and interphase after surface spreading and critical point drying and as thin sections after fixation and embedding. Chromosomes and interphase nuclei were composed of irregularly coiled fibers measuring about 200 to 300 Å in width. Thinner and straighter chromosome fibers were produced by stretching or NaCl extraction. Thin sections of metaphase chromosomes showed 200 Å wide granular outlines containing regularly coiled 70–80 Å wide fibrils. These smaller substructures appeared hollow with a 20 Å thick electron dense wall. The possible arrangement of the DNA molecule as a tertiary coil in the chromosome fiber of living cells is suggested.

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Zusammenfassung Menschliche Chromosomen und-Fäden in Meta- und Interphase wurden als Totalpräparate nach Oberflächenspreitung und Kritischer-Punkt-Trocknung und als Dünnschnitte nach Fixierung und Einbettung mit dem Elektronenmikroskop untersucht. Metaphase-Chromosomen und Interphase-Kerne fanden sich als aus unregelmäßig gewundenen Fäden von 200–300 Å Dicke bestehend. Durch Dehnung und NaCl-Extraktion wurden diese Chromosomenfäden dünner und gerader. Dünnschnitte der Metaphase-Chromosomen zeigten 200 Å breite granuläre Fadenkonturen, die regelmäßig gewundene, spiralig angeordnete 70–80 Å breite Fibrillen enthielten. Diese fibrillären Unterstrukturen erschienen hohl mit einer 20 Å dicken elektronendichten Wand. Die mögliche Anordnung des DNA-Moleküls als Tertiärschraube im Chromosomenfaden lebender Zellen wird diskutiert.

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Supported by a grant (La 185/3) of the Deutsche Forschungsgemeinschaft.     

Cytological map of human Y chromosome

The cytological map of human Y chromosome is investigated at high resolution using Q-banding technique. There was no segmentation of the short arm which is in disagreement with current International Nomenclature. Also, the band q11.22 was not seen in the long arm using different individuals with variable size of Y chromosome. Furthermore, the length variation of nonfluorescent segment is also noted which was not suggested by the nomenclature.