Mapping of the transferrin gene in laboratory rats, mice and man by direct in situ hybridization

Mapping of the gene coding for transferrin was carried out in metaphase chromosomes from bone marrow of laboratory mice and rats as well as from PHA-stimulated human lymphocytes using direct in situ hybridization technique. Plasmid pRTf-17 carrying the insert of rat transferrin cDNA was nick-translated with [125I]dCTP and used as a specific hybridization probe. The total number of silver grains and their distribution along differentially stained chromosomes were determined in 464 metaphase plates (114, 263 and 87 from rat, mouse and man, respectively). The data obtained enable us to assign transferrin gene to chromosome 3 in human and chromosome 9 in mouse. For the first time, the rat transferrin gene was localized on chromosome 7. The most probable sites of transferrin gene localization are 7q31-34, 9F1-3 and 3q21 in rat, mouse and human chromosomes, respectively.     

Clustering of Human Chromosome Fragments on the Mouse Genome by Chromosome-Mediated Gene Transfer

Pieces of metaphase chromosomes prepared from mouse cells containingneo-tagged human chromosome 7 were transferred to mouse cellswith calcium phosphate to isolate G418-resistant clones. FISHanalysis revealed that the majority of them contained humanDNA at a single site on their genome. These transformants containedSTS markers mapped to various regions of chromosome 7. It isthus suggested that pieces of human chromosomes tend to assembleand integrate on the mouse genome.     

Lack of detectable kinetochores on some chromosomes in mouse x human hybrids.

When treated with an anti-kinetochore antibody present in the sera of scleroderma (var. CREST) patients, most chromosomes exhibit kinetochore dots at the position of the centromere. In this paper we report that some chromosomes in the mouse x human somatic cell hybrid fail to show these dots. In the early passages in a hybrid, HYG-1, the frequency of such chromosomes was higher (0.85%) than in later passages (0.45%) studied after five months of continuous culturing. In parallel, the mean number of human chromosomes in the hybrid also dropped. The somewhat hypodiploid parental cell lines, when similarly treated, showed only a rare chromosome without kinetochore dots. Immunoblots of the proteins showed that the sera used for kinetochore detection recognized all major centromere proteins (CENPs). Electron microscopy of some offlying metaphase chromosomes in another hybrid, HR61, exhibited a lack of trilamellar kinetochores. This study suggests that akinetochoric chromosomes might provide a novel mechanism responsible for chromosome loss and genesis of aneuploidy. In early passages, some cells in the hybrid showed detached kinetochores. These autonomous kinetochores could be seen in clusters and involved some mouse chromosomes also. Potential significance of these autonomous kinetochores in generating compound centromeres is discussed.     

小鼠中期染色体制备方法探讨

目的:寻找小鼠细胞中期染色体制备过程中理想的取材方法和低渗的最佳时间,以及制片时滴片的最佳高度。方法:根据以往小鼠染色体制备方法的基本步骤,在取材方面进行对比,同时分别设置4个加入低渗液的时间和4个滴片高度进行试验对比。结果:各低渗时间段和各滴片高度所制备的结果存在一定的差异性,制备成功率和良好率最高的低渗时间为25 min,滴片高度为20 cm。结论:取材骨髓比较容易和简洁制备中期染色体,最佳低渗时间为25 min,最佳滴片高度为20 cm。     

Transfer of the herpes simplex thymidine kinase gene from human cells to mouse cells by means of metaphase chromosomes.

Thymidine kinase (TK)-deficient human cells were infected with ultraviolet light-inactivated Herpes simplex virus type 1, and "transformed" cells that expressed Herpes TK activity were isolated. Purified metaphase chromosomes were isolated from the transformed human line and incubated with TK-deficient mouse cells. TK+ cells were selected, and it was shown that these cells were gene transferents which expressed Herpes TK activity, identical to that found in the transformed human cells. The gene transferents contained no intact human chromosomes. When removed from selective pressure, the gene transferents rapidly lost the TK+ phenotype. However, upon continued growth in nonselective medium, a subpopulation in which the TK+ phenotype had become more stabilized appeared. These results suggest that the Herpes gene for thymidine kinase has integrated into the genome of the HSV-transformed human cells and that it can be transferred to other cells by means of purified metaphase chromosomes.     

A Simple Staining Method for the Analysis of Meiotic Metaphase II Divisions in the Male Mouse

During an investigation into the effects of X rays on meiosis in the male mouse (Szemere and Chandley 1975) a staining technique was required that would enable us to make an accurate analysis of dyads² at metaphase II. Not only were we interested in analysing chromosomal aberrations at this stage, but we also wished to identify with confidence the X and Y chromosomes, and to establish accurate counts of dyad numbers. Conventional staining with carbol fuchsin (Can and Walker 1961) provided adequate means for recognizing sex chromosomes, but centromere positions could not be identified and little morphological detail of autosomal dyads could be discerned. Staining by the BSG barium hydroxide/saline/Giemsa technique (Sumner 1972) as modified for use on meiotic cells of the mouse (Chandley and Fletcher 1973) gave excellent staining of centric heterochromatin, but dyad arms were often pale and indistinct. Other centromere staining methods for murine meiotic cells (Hsu, Cooper, Mace and Brinkley 1971, Polani 1972), gave unsatisfactory results in our hands. By combining carbol fuchsin staining with the BSG centromere staining technique, we have been able to produce a simple and quick technique which gives excellent staining of centromeres, easy identification of X and Y chromosomes and good staining of dyad arms at metaphase II. The technique has also been applied successfully to other meiotic stages of the mouse and to human somatic metaphase chromosomes.     

STUDIES ON THE ISOLATION OF METAPHASE CHROMOSOMES

A method for the isolation of metaphase chromosomes from mouse L1210 leukemia cells has been developed. Cells, arrested at metaphase with colchicine, were exposed to hypotonic solution and the pH was then adjusted to 5.6 to stabilize the chromosomes. The metaphase figures were subsequently disrupted and the chromosomes isolated by a series of differential centrifugations in sucrose. The isolated chromosomes were well preserved, as judged by morphological criteria. The effect of various enzymes and chemical agents on the isolated chromosomes was studied. Chymotrypsin, trypsin, and deoxyribonuclease caused a marked disintegration of the chromosomes, whereas treatment with pepsin and ribonuclease induced no significant morphological alterations.     

Mapping of the dopa decarboxylase gene to the 11A band of the murine genome.

A human DOPA decarboxylase (DDC) cDNA probe of 747 base pairs has been used to map the DDC gene by in situ hybridization on mouse metaphase chromosomes. This result indicates that the gene is located on band 11A, near the erythroblastosis oncogene B (erb b) locus. This provides evidence for a synteny group on mouse chromosome 11 and human chromosome 7.     

In situ hybridisation localises the gene for the major intrinsic protein of eye lens fibre cell membranes to human chromosome 12q14.

The gene encoding the major intrinsic protein (MIP) of eye lens fibre cell membranes has been localised to human chromosome 12q14 by in situ hybridisation of a cDNA for rat MIP to G-banded metaphase chromosomes. The human MIP gene maps within a conserved region of synteny with mouse Chromosome 10.     

Localisation of the gene for the major intrinsic protein of eye-lens-fibre cell membranes to mouse chromosome 10 by in situ hybridisation.

The gene encoding the major intrinsic protein (Mip) of eye-lens-fibre cell membranes has been assigned to region D1 of mouse Chromosome 10 by in situ hybridisation of a cDNA for rat MIP to G-banded metaphase chromosomes. The mouse Mip gene maps within or near to a segment homoeologous with human chromosome 12q and may be linked to the Cat locus at the distal end of mouse Chromosome 10.     

Rapid analysis of mouse-hamster hybrid cell lines by in situ hybridization

In situ hybridization techniques for analyzing the murine DNA complement of mouse-hamster hybrid cells are described. Total genomic mouse DNA is labeled with biotin and hybridized without suppression to metaphase spreads from a mouse-hamster hybrid line containing the mouse fusion chromosome X12. Detection via fluorochrome-conjugated avidin reveals mouse chromosomal DNA with high sensitivity and permits the identification of both normal and aberrant murine chromosomes. Conversely, biotinylated total genomic DNA from a hybrid line can be used as a probe on normal mouse metaphase spreads if suppression techniques are employed, facilitating the analysis of mouse chromosomes present in the hybrid line.     

A Simple Staining Method for the Analysis of Meiotic Metaphase II Divisions in the Male Mouse

During an investigation into the effects of X rays on meiosis in the male mouse (Szemere and Chandley 1975) a staining technique was required that would enable us to make an accurate analysis of dyads² at metaphase II. Not only were we interested in analysing chromosomal aberrations at this stage, but we also wished to identify with confidence the X and Y chromosomes, and to establish accurate counts of dyad numbers. Conventional staining with carbol fuchsin (Can and Walker 1961) provided adequate means for recognizing sex chromosomes, but centromere positions could not be identified and little morphological detail of autosomal dyads could be discerned. Staining by the BSG barium hydroxide/saline/Giemsa technique (Sumner 1972) as modified for use on meiotic cells of the mouse (Chandley and Fletcher 1973) gave excellent staining of centric heterochromatin, but dyad arms were often pale and indistinct. Other centromere staining methods for murine meiotic cells (Hsu, Cooper, Mace and Brinkley 1971, Polani 1972), gave unsatisfactory results in our hands. By combining carbol fuchsin staining with the BSG centromere staining technique, we have been able to produce a simple and quick technique which gives excellent staining of centromeres, easy identification of X and Y chromosomes and good staining of dyad arms at metaphase II. The technique has also been applied successfully to other meiotic stages of the mouse and to human somatic metaphase chromosomes.     

Asymmetric decondensation of the L cell heterochromatin by Hoechst 33258

A benzimidazole derivative, Hoechst 33258 can induce decondensation of constitutive heterochromatin in the mouse derived L cell chromosomes when the compound is given in sufficiently high concentration (40 micrograms/ml) to the L cell culture. Hoechst 33258 at low concentration (1 micrograms/ml, 16 h) cannot produce this effect on L cell chromosomes. Bromodeoxyuridine (BUdR) incorporation for one cell cycle simultaneous with the Hoechst 33258 treatment at low concentration could decondense heterochromatin segments in metaphase chromosomes. The heterochromatin decondensation, however, was asymmetric; it was observed only on one chromatid and the other of a chromosome remained in condensed state. The observation of asymmetric decondensation of heterochromatin by Hoechst 33258 after BUdR incorporation for one cell cycle, the association of A-T rich satellite DNA to mouse heterochromatin, and available data on the specific binding of Hoechst 33258 to A-T base pairs of DNA and on the higher affinity of the compound to BUdR substituted DNA than to ordinary DNA implied that the binding of Hoechst 33258 molecules to A-T rich satellite DNA is the cause of heterochromatin decondensation.     

Cytogenetic Analysis of Spermatozoa in a Patient with Chromosome Constitution 45,X/46,X,r(Y) by Intracytoplasmic Injection into Mouse Oocytes

The chromosome set of human spermatozoa was studied by intracytoplasmic injection into mouse oocytes. A total of 85 metaphase plates of male pronuclei of a patient with chromosome constitution 45,X/46,X,r(Y) and 108 metaphase plates of patients with normal sperm parameters (control group) were examined. The ratio between X- and Y-bearing chromosomes in the 45,X/46,X,r(Y) patient and in the control group did not differ from 1 : 1. A significant increase in the rates of diploidy, hypoploidy, hyperploidy of sex chromosomes, and chromosome structure rearrangements in spermatozoa of the patient in comparison with spermatozoa in the control group was recorded.     

Cytogenetic analysis of spermatozoa in a patient with chromosome constitution 45,X/46,X,r(Y) by intracytoplasmic injection into mouse oocytes

The chromosome set of human spermatozoa was studied by intracytoplasmic injection into mouse oocytes. A total of 85 metaphase plates of male pronuclei of a patient with chromosome constitution 46,X,r(Y)/45,X and 108 metaphase plates of patients with normal sperm parameters (control group) were examined. The ratio between X- and Y-bearing chromosomes in the 46,X,r(Y)/45,X patient and in the control group did not differ from 1:1. A significant increase in the rates of diploidy, hypoploidy, hyperploidy of sex chromosomes, and chromosome structure rearrangements in spermatozoa of the patient in comparison with spermatozoa in the control group was recorded.     

A new method for the preparation of metaphase chromosomes for flow analysis

A new method for the preparation of metaphase chromosomes for flow analysis has been evaluated. It has been shown that this method, which involves detergent lysis of metaphase cells and polyamines to stabilize the DNA, yields lower coefficients of variation and background levels in the DNA histograms than is currently obtained by hexylene glycol based methods. A conventional flow cytometer (FACS-II) has been used to resolve the human karyotype into about 14 peaks after ethidium bromide staining and excitation with a relatively low level of illumination (0.4 W at 488 nm). Flow karyotypes have also been obtained from suspension cell lines, in particular from the mouse cell line, Friend 707/B10. The only disadvantage of this method is that the chromosomes are highly condensed and therefore banding studies on sorted chromosomes may not be possible.     

Do individual allocyclic chromosomes in metaphase reflect their interphase domains?

Summary Individual S phase allocyclic chromosomes have been analyzed in Bloom syndrome lymphocytes, in cells with an r(9), and in hypotetraploid Ehrlich mouse ascites cells treated with 1-methyl-2-benzyl hydrazine. On the basis of the following observations, we conclude that such chromosomes more or less reflect their domains in interphase: (1) The S phase allocyclic chromosomes have the same structure as S phase prematurely condensed chromatin (PCC) in fused cells; in other words they form limited areas of chromatin dots; (2) the allocyclic chromosome is the only chromosome in a metaphase plate which synthesizes DNA simultanneously with interphase nuclei; (3) the size of the allocyclic chromosomes is related to the size of the corresponding metaphase chromosome; and (4) the S phase allocyclic chromosomes resemble closely the chromosome domains in interphase made visible with biotinylated human DNA. A variety of evidence shows that most allocyclic chromosomes are simply left behind in their cycle, which presumably is caused by a deletion or inactivation of a hypothetical coiling center situated on each chromosome arm.     

High resolution G-banded chromosomes of the mouse

High resolution G-banded mouse chromosomes were prepared using an actinomycin D and acridine orange pretreatment protocol, resulting in late prophase mouse chromosomes which reveal over twice the number of bands as compared with mid metaphase. These elongated chromosomes, described here in detail and used to construct a precise schematic representation of the late prophase banding patterns, should be generally useful in high resolution mouse chromosome analysis.     

The human aldose reductase gene maps to chromosome region 7q35

Summary The human aldose reductase (AR) gene has been mapped to chromosome 7 using the polymerase chain reaction to specifically amplify the human AR sequence in hamster/human hybrid DNA and also in mouse/ human monochromosome hybrids. The assignment to chromosome 7 was confirmed by in situ hybridisation to human metaphase chromosomes using a novel, rapid hybridisation, method giving a regional localisation at 7q35.