Mouse Metaphase Research Articles

Selective digestion of mouse metaphase chromosomes.

Metaphase chromosomes prepared from colcemid-treated mouse L929 cells by non-ionic detergent lysis exhibit distinct heterochromatic centromere regions and associated kinetochores when viewed by whole mount electron microscopy. Deoxyribonuclease I treatment of these chromosomes results in the preferential digestion of the chromosomal arms leaving the centromeric heterochromatin and kinetochores apparently intact. Enrichment in centromere material after DNase I digestion was quantitated by examining the increase in 10,000 X g pellets of the 1.691 g/cc satellite DNA relative to main band DNA. This satellite species has been localized at the centromeres of mouse chromosomes by in situ hybridization. From our analysis it was determined that DNase I digestion results in a five to six-fold increase in centromeric material. In contrast to the effect of DNase I, micrococcal nuclease was found to be less selective in its action. Digestion with this enzyme solubilized both chromosome arms and centromeres leaving only a small amount of chromatin and intact kinetochores.

Non-histone protein associated with centromeric heterochromatin in the mouse chromosome

Distribution of protein in cytological preparations of the mouse metaphase chromosome was studied using protein-specific fluorochroming with dansyl chloride. Dansyl-fluorescence was relatively uniform along the axis of untreated chromosomes. After treatment of air-dried chromosome preparations with 0.2 N HCl, dansyl-fluorescence in chromosomes was remarkably reduced; it exhibited, however, bright fluorescent blocks at centromeric regions in almost all chromosomes. The same situation was observed in the male meiotic chromosomes at diakinesis; the Y chromosome seemed to lack bright dansyl-fluorescent block at the centromere after 0.2 N HCl extraction. The bright dansyl-fluorescent blocks in chromosomes after HCl or NaCl treatment occupied the same regions as C-bands. These observations suggested that centromeric constitutive heterochromatin in the mouse chromosome was associated with particular non-histone protein.

Reverse banding on chromosomes produced by a guanosine-cytosine specific DNA binding antibiotic: olivomycin.

Characteristic reverse fluorescent banding patterns (R bands) on human, bovine, and mouse metaphase chromosomes are produced by treating chromosome preparations directly with olivomycin. With the DNA in solution, the repeating polymer poly[d(G-C)] - poly[d((G-C)] (where G is guanine and C is cytosine) enhanced the fluorescence of olivomycin, while the antibotic fluorescence was not affected by the alternating polynucleotide poly]d(A-T)] - poly[d(A-T)] (where A is adenine and T is thymine). Calf thymus DNA, with an intermediate G-C content of about 40 percent, showed a smaller fluorescence enhancement in the presence of olivomycin as was observed for the synthetic polynucleotide poly[d(G-C)] - poly [d(G-C)]. The closely related antibiotic chromomycin A3 showed the same results as were obtained with olivomycin either in the solution interaction with specific DNA's or with the netaphase chromosome preparations. The production of R bands by these G-C-specific DNA binding antibiotics lends credence to the suggestion that the arrangement of the nucleotide sequences along the chromosome is a primary determinant for the appearance of fluorescent bands.

The structural organization of mouse metaphase chromosomes.

The binding of highly purified anti-nucleoside antibodies to mouse (Mus musculus) metaphase chromosomes was studied by an immunofluorescence technique. The chromosomal DNA was denatured by one of two selective denaturation procedures because these antibodies reacted with single stranded but not native DNA. After ultraviolet irradiation (UV), which produced single stranded regions primarily in AT rich DNA, the binding of antiadenosine (anti-A) produced a pattern of fluorescent bands similar to that produced by quinacrine (Q-bands). Additional foci of bright fluorescence were observed at the centrometric (C-band) regions, which are known to contain AT rich satellite DNA. After photooxidation, which produced single stranded regions in GC rich DNA, the binding of anti-A produced a fluorescent banding pattern similar to the R-banding pattern seen after thermal denaturation and staining with coriphosphine O. After photooxidation, R-band patterns were also obtained with anti-cytidine (anti-C) and anti-5-methylcytidine (anti-M). After either UV irradiation or photooxidation, anti-M, but not anti-C, showed intense binding to the C-band regions of mouse chromosomes. — These findings led to the following conclusions: (1) Antibody banding patterns reflect the presence of a class of AT rich, GC poor DNA in chromosome regions which show bright quinacrine fluorescence and in the regions that contain the AT rich satellite DNA. (2) The alternate, quinacrine dull regions contain a relatively GC rich class of DNA which appears to be more highly methylated than the AT rich DNA in the Q-bright bands, but not the AT rich satellite DNA in the Q-dull C-bands. (3) 5-Methylcytosine residues occur in a sequence of mouse satellite DNA that contains both adjacent pyrimidines and guanine residues. The basic repeating unit of mouse satellite DNA is known to contain the sequence 5′-GAAAAATGA-3′ (Biro et al., 1975). Therefore, assuming the antibodies used could detect single bases in denatured DNA, the methylated sequence in mouse satellite DNA \({\text{could be }}\begin{array}{*{20}c} {5\prime - {\text{A M G AAAAA T GA - 3}}\prime } \\ {3\prime - {\text{T G M TTTTT A C T - 5}}\prime } \\ \end{array} {\text{ or }}\begin{array}{*{20}c} {5\prime - {\text{G AAAAA T G A M G AA - 3}}\prime } \\ {3\prime - {\text{C T T T T T AC T G M TT - 5}}\prime } \\ \end{array} .\)

A method for discriminating murine and human chromosomes in somatic cell hybrids

Treatment of cultured mouse cells with the benzimidazole compound “33258 Hoechst” induces an enlargement of the pericentric area of most mouse metaphase chromosomes. The drug, however, has no effect on human chromosomes. Therefore, pretreatment of mouse-man somatic cell hybrids with the compound allows easy discrimination of murine and human chromosomes in the metaphase preparations.

The use of antinucleoside antibodies to probe the organization of chromosomes denatured by ultraviolet irradiation

Ultraviolet irradiation of methanol: acetic acid-fixed human and mouse metaphase chromosomes rendered them capable of binding antibodies specific for purine or pyrimidine bases. Since these antibodies react with single-stranded but not with native DNA, our results indicate that UV irradiation generated single-stranded regions in chromosomal DNA. Using an indirect immuno-fluorescence technique to detect antibody binding, highly characteristic, nonrandom patterns of antibody binding were observed. Antibodies to adenosine (anti-A) and thymidine (anti-T) produced identical patterns of binding which in most respects matched the chromosome banding patterns produced by quinacrine. However, additional foci of intense fluorescence were seen in the paracentromeric regions of constitutive heterochromatin on chromosomes 1, 9 and 16, regions which had been shown by in situ DNA-RNA hybridization to be the locations of AT-rich human satellite DNA. Antibodies to cytidine also bound to the same region of chromosome 9. In mouse chromosome preparations, both anti-A and anti-T produced bright fluorescence of the region containing centromeric heterochromatin, which had been shown to be the location of the AT-rich satellite DNA of this species.

Mechanisms of chromosome banding: II. Evidence that histones are not involved

C-and G-banding in mouse metaphase chromosomes is unaffected by exposure of the chromosomes to 0.2 N HCl for 4 h. Electrophoretic studies indicate that this is adequate to completely remove the histones from fixed and dried chromatin thus indicating that histones are not involved in C- or G-banding.