The deoxyribonucleoprotein complex from the archaebacterium Methanosarcina barkeri: characterization and ultrastructural localization of the chromosomal protein MC1

The Methanosarcina barkeri protein, methanogen chromosomal protein 1 (MC1), a basic polypeptide of 93 amino acid residues, has been localized in the DNA-rich areas on immunolabelled bacterial cryosections revealed with the protein A – gold method. This localization strongly supports the previous studies concerning the association of the protein MC1 with the DNA in vivo. In the M. barkeri deoxyribonucleoprotein complex, the protein MC1 accounts for about 90% of the total proteins, and the protein MC1 so DNA ratio (by weight) is equal to 0.11. Staphylococcal nuclease digestion data and microscopic observations have failed to reveal the presence of repeating structural units and suggest that the protein MC1 is unevenly distributed along the DNA.

STUDIES ON DEOXYRIBONUCLEOPROTEIN IN LEUKOCYTES AND RELATED CELLS OF MAMMALS VII. THE FAST GREEN HISTONE CONTENT OF RABBIT LEUKOCYTES AFTER HYPOTONIC TREATMENT

Peripheral leukocytes of rabbit were subjected to the following treatments: (1) 3 parts blood to 2 parts water; (2) 0.95% sodium citrate; (3) Ohnuki's medium. Treated and control leukocytes were then smeared, fixed in 9:1 methanol-commercial formalin and stained for histones according to Alfert and Geschwind. Cytophotometry was carried out with the Barr and Stroud integrating microdensitometer. The cells treated with water increase their dye content by 50%; this increase is less marked (30%) in the cells incubated in Ohnuki's mixture, while there is a 15% decrease in case of citrate. The spread of values "between cells" is larger than the values found with the Feulgen reaction because of factors other than chromatin compaction. (1) There are fluctuations in staining intensity in different areas of the same slide. (2) Nonhistone substrates take the dye, such as hemoglobin of erythrocytes, cytoplasm and specific granules of heterophil granulocytes. (3) Apparently, a certain amount of histones is lost when nuclei swell in hypotonic citrate. It is concluded that, although histone dye binding behaves in a manner similar to Feulgen with respect to changes in the physical state of the deoxyribonucleoprotein complex, the quantitation of fast green histone is a less reliable method for the study of these changes.

Changes in The Cytochemical Properties Of Erythrocyte Nuclei Reactivated By Cell Fusion

When the nucleus of a chick erythrocyte is introduced into the cytoplasm of a HeLa cell it resumes the synthesis of RNA and DNA. This reactivation of the red cell nucleus is associated with an increase in volume and with changes in nuclear composition. These changes have now been studied by quantitative cytochemical techniques. During the process of reactivation the dry mass of the erythrocyte nucleus shows a marked increase which takes place largely before the replication of the DNA begins. Within 24 h of cell fusion, some erythrocyte nuclei already contain an increased amount of DNA, and 48 h after fusion many of them contain twice the normal diploid amount, thus indicating that they have replicated their DNA completely. The physical properties of the nuclear deoxyribonucleoprotein complex also change. The ability of the nuclear chromatin to bind acridine orange increases 4- to 5-fold well before the synthesis of DNA begins; and changes in the melting profile of the deoxyribonucleoprotein suggest that its structure is loosened. This view is also supported by the observation that the reactivity of the erythrocyte nuclei to the Feulgen stain is altered during the early stages of reactivation.

STUDIES ON DEOXYRIBONUCLEIC ACID IN LEUKOCYTES AND RELATED CELLS OF MAMMALS VI. THE FEULGEN-DEOXYRIBONUCLEIC ACID CONTENT OF RABBIT LEUKOCYTES AFTER HYPOTONIC TREATMENT

Peripheral blood of rabbit was subjected to the following hypotonic treatments in addition to controls: (1) 3:2 blood or plasma-distilled water; (2) 0.2 M sucrose; (3) 0.95% sodium citrate; (4) Ohnuki's medium ( Nature (London) 208: 916. 1965). The cells were smeared, fixed in methanol-acetic-formalin and stained by means of the Feulgen reaction. Cytophotometry was carried out in the Barr and Stroud integrating microdensitometer. The results show that the cells subjected to hypotonic media increase their chromatic load, the mean values being 7-12% higher than the controls. These results reinforce previous suggestions that any change in the physical state of deoxyribonucleoprotein molecules brings concomitant changes in the reactivity of their dye-binding sites and, moreover, that this increase is correlated with (1) diffusion of the deoxyribonucleoprotein complex and (2) increase of the mean nuclear size. Therefore, under the conditions used, the more diffuse the nuclear content, the higher the apparent deoxyribonucleoprotein value.