Compact form of SV40 viral minichromosome is resistant to nuclease: possible implications for chromatin structure.

We report two new findings bearing on the "supranucleo-somal" level of the structure of the Simian Virus 40 minichromosome. I) Isolated SV40 minichromosome which contains all five histones including HI/I/ exists in solution under approximately physiological ionic conditions as a compact roughly spherical particle approximately 300 A in diameter which is capable of fitting within the virus capsid. In spite of such a compact conformation of the minichromosome individual nucleosomes can be readily visualized within the particle. Compact state of SV40 minichromosome depends on both the presence of histone HI and maintenance of approximately physiological ionic strength of solution (micron approximately 0.15). Removal of HI results in a conversion of the compact minichromosomes into an extended (circular beaded) structure. 2) The compact form of the SV40 minichromosome in contract to its circular beaded form is virtually completely resistant to staphylococcal nuclease, strongly suggesting that in particular nuclease-sensitive parts of the internucleosomal DNA regions are not exposed on the outside of the compact SV40 minichromosome. On the other hand, DNase I which is known to attack both inter-and intranucleosomal DNA in the chronatin /2,3/ readily digests the compact form of the SV40 minichromosome. Possible models of the compact minichromosome and implications for higher order structures of the cellular chromatin are discussed.     

Revision of SAT chromosome number in Triticum monococcum with respect to nucleolar organizers activity

Summary Six varieties of Triticum monococcum were analysed by means of the nucleolar test; i.e., estimation of the maximum number of primary nucleoli per nucleus. All of the varieties exhibited 4 primary nucleoli in telophase and early interphase. Following detailed karyological analysis four SAT chromosomes in all six karyotypes were found in accordance with the maximum nucleolar number. Secondary constrictions and microsatellites were localised on the short arms of chromosome pairs 3 and 5. A new order of the chromosomes in the idiogram of Tr. monococcum is proposed.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Dynamics of viscoelastic spherical membranes—the balloon model of the alveolus

Double-valued pressure-volume relationships in dynamic conditions for spherical membranes, modelling the lung alveoli, were obtained at small deformations. This hysteretic behavior was considered to be produced by at least three independent mechanisms: (1) the lung parenchyma exhibits viscoelastic properties; (2) the lung surface film, independent of the tissue, exhibits viscoelastic properties and (3) the pressure acting on the inner membrane surface depends on the rate of the alveolus volume change, due to the air viscous resistance in the bronchial tree. In each case, the maximum volume change, the hysteresis loop area, the tilt angle of the hysteresis loop and the relaxation time of the system were calculated. The results show pronounced hysteresis at normal breathing due to the air viscous resistance and smaller one due to the tissue and surface viscoelastic properties. In quasistatic conditions the values of the surface viscoelasticity and the tissue viscoelasticity effects are comparable or different, depending on the concrete external conditions. Comparison with the available experimental data is discussed in detail.     

Mobile dispersed genetic elements in eukaryotes and their possible relationship to carcinogenesis

During last three years, the mobile dispersed genetic elements (mdg) were isolated from the genome of Drosophila melanogaster, yeasts and mammals. According to a number of their properties, mdg elements are quite similar to endogenous pro-retroviruses. It is known that in many cases oncogeneity of retroviruses depends on the incorporation of the certain host genes (potential oncogenes) into the viral genome. We suggest that in some cases mdg elements could entrap the potential oncogenes in the course of transposition. As a result, oncogenes become uncontrollable by host regulatory systems and may induce cell transformation. Another possible mechanism underlying switch off of the gene responsible for differentiation control may be mdg transposition to a region in close vicinity of the gene. As transposition of mdg elements seems to occur rather often, they may be regarded as one of the most important factors of genome rearrangements leading to cell transformation.     

Long double-stranded sequences (dsRNA-B) of nuclear pre-mRNA consist of a few highly abundant classes of sequences: evidence from DNA cloning experiments.

DNA preparations from about hundred randomly selected clones containing mouse DNA fragments were screened for the existence of sequences complementary to long double-stranded regions of pre-mRNA able to snap back after melting (dsRNA-B). Many clones containing such sequences were found. The cloned sequences can be subdivided into three groups: (1) those complementary to about a half (at least to 30-40%) of the total dsRNA, designated as sequences B1; (2) those complementary to a part of sequence B1; and (3) sequences complementary to about a quarter (at least to 15%) of the total dsRNA referred to as sequence B2. The size of DNA sequence complementary to dsRNA is about 400 base pairs.Melting experiments with hybrids show that the members of B1 family are very similar if not identical, while the divergence among B2 sequences is higher, but still the number of substitutions does not exceed 9% of bases. Thus, the major part of dsRNA-B consists of a small number of highly abundant sequences as was suggested earlier on the basis of renaturation kinetics /1-3/. Sequences B1 and B2 are represented by many copies in the mouse genome and in pre-mRNA, and many of them probably do not form hairpin-like structures.