Menashe Marcus

Mapping of DNAase I sensitive regions on mitotic chromosomes.

We have shown that in fixed mitotic chromosomes from female G. gerbillus cells the inactive X chromosome is distinctly less sensitive to DNAase I than the active X chromosome, as demonstrated by in situ nick translation. These results indicated that the specific chromatin conformation that renders potentially active genes sensitive to DNAase I is maintained in fixed mitotic chromosomes. We increased the sensitivity and accuracy of in situ nick translation using biotinylated dUTP and a specific detection and staining procedure instead of radioactive label and autoradiography and now show that in both human and CHO chromosomes, the DNAase I sensitive and insensitive chromosomal regions form a specific dark and light banding pattern. The DNAase I sensitive dark D-bands usually correspond to the light G-bands, but not all light G-bands are DNAase I sensitive. Identifiable regions of inactive constitutive heterochromatin are in a DNAase I insensitive conformation. Our methodology provides a new and important tool for studying the structural and functional organization of chromosomes.

In situ nick-translation distinguishes between active and inactive X chromosomes

Template-active regions of chromatin are structurally distinct from nontranscribing segments of the genome. Recently, it was suggested that the conformation of active genes which renders them sensitive to DNase I may be maintained even in fixed mitotic chromosomes. We have developed a technique of mitotic cell fixation and DNase I-directed nick-translation which distinguishes between active and inactive X chromosomes. We report here that Gerbillus gerbillus (rodent) female cells contain easily identified composite X chromosomes each of which includes the original X chromosome flanked by two characteristic autosomal segments. After nick-translation the active X chromosome in each cell is labelled specifically in both the autosomal and X-chromosomal regions. The inactive X chromosome is labelled only in the autosomal regions and in a small early replicating band within the late replicating ‘original X’ chromosome. Our technique opens the possibility of following the kinetics of X-chromosome inactivation and reactivation during embryogenesis, studying active genes in the inactive X chromosome and mapping tissue-specific gene clusters.

DNA hypomethylation causes an increase in DNase-I sensitivity and an advance in the time of replication of the entire inactive X chromosome

SummaryWe have examined the effect of 5-azacytidine (5-aza-C) induced hypomethylation of DNA on the time of replication and DNase I sensitivity of the X chromosomes of female Gerbillus gerbillus (rodent) lung fibroblast cells. Using in situ nick translation to visualise the potential state of activity of large regions of metaphase chromosomes we show that 5-aza-C causes a dramatic increase in the DNase-I sensitivity of the entire inactive X chromosome of female G. gerbillus cells and this increase in nuclease sensitivity correlates with a large shift in the time of replication of the inactive X chromosome from late S phase to early S phase. These effects of 5-aza-C on the inactive X chromosome are associated with a 15% decrease in DNA methylation. Our results indicate that DNA methylation concomitantly affects both the time of replication and the chromatin conformation of the inactive X chromosome.

DNase I sensitivity in facultative and constitutive heterochromatin

In situ nick translation allows the detection of DNase I sensitive and insensitive regions in fixed mammalian mitotic chromosomes. We have determined the difference in DNase I sensitivity between the active and inactive X chromosomes inMicrotus agrestis (rodent) cells, along both their euchromatic and constitutive heterochromatic regions. In addition, we analysed the DNase I sensitivity of the constitutive heterochromatic regions in mouse chromosomes. InMicrotus agrestis female cells the active X chromosome is sensitive to DNase I along its euchromatic region while the inactive X chromosome is insensitive except for an early replicating region at its distal end. The late replicating constitutive heterochromatic regions, however, in both the active and inactive X chromosome are sensitive to DNase I. In mouse cells on the other hand, the constitutive heterochromatin is insensitive to DNase I both in mitotic chromosomes and interphase nuclei.

Pattern of condensation of mouse and Chinese hamster chromosomes in G2 and mitosis of 33258-Hoechst-treated cells

Abstract The fluorochrome 33258-Hoechst which binds to DNA and preferentially to A-T-rich regions, inhibits drastically the condensation of the centromeric heterochromatic regions in mouse cell lines. Condensation of all other regions of the chromosomes is also inhibited to some extent. The kinetics of condensation-inhibition of the C-heterochromatin indicates that these regions are being condensed at specific time intervals in the G2 period with a specific order of condensation. The C-heterochromatic regions of mouse chromosomes nos. 9, 12, 14, 15 and 16 condense late in G2 and complete their condensation about 30 min before metaphase. Condensation in G2 of Chinese hamster chromosomes is also inhibited by 33258-H treatment.

Condensation-inhibition by 33258-Hoechst of centromeric heterochromatin in prematurely condensed mouse chromosomes☆

Abstract The phenomenon of premature chromosome condensation has been applied to study the kinetics of condensation-inhibition exerted by the fluorochrome 33258-Hoechst (33258-H) on the centromeric heterochromatic regions of mouse chromosomes. Asynchronous mouse A-9 cells in culture were fused with mitotic HeLa cells in the presence of 33258-H. Pronounced condensation-inhibition of the c-heterochromatin was observed in prematurely condensed early G2, S and late G1 chromosomes in the 33258-H-treated cells. It is concluded that the c-heterochromatic regions begin to condense quite early in G2, decondense again late in G1 and remain decondensed in the S phase.

The pleiotropic effects of 33258-Hoechst on the cell cycle in Chinese hamster cells in vitro.

Abstract The fluorochrome 33258-Hoechst which binds to double-stranded DNA (dsDNA) has been previously shown to inhibit in several mammalian cell cultures the condensation of chromosomes in phase G2 and early mitosis. We have now found that this drug affects the cell cycle of Chinese hamster cells grown in vitro in several other ways. In cells treated with the drug, phase G2 is prolonged, the rate of DNA replication is drastically reduced and the cells are arrested most probably at very late S phase.

Condensation of all human chromosomes in phase G2 and early mitosis can be drastically inhibited by 33258-Hoechst treatment

SummaryCondensation of human chromosomes in phase G2 and early mitosis is inhibited by the fluorochrome 33258-Hoechst. This inhibitory effect is most apparent in primary diploid fibroblasts and lymphoblasts and least pronounced in peripheral blood lymphocytes. Condensation of the human Y chromosome, which contains a large heterochromatic region rich in A-T base pairs, is drastically inhibited by 33258-Hoechst treatment of fibroblasts and lymphoblasts. The difference in sensitivity of human chromosomes in different cell types to 33258-Hoechst probably reflects differences in the cell-membrane permeabilities to 33258-Hoechst.

The metabolic pathway of glutamate in escherichia coli K-12

Abstract Mutants unable to grow on glutamate as the sole source of carbon and energy, isolated from glutamate-utilizing Escherichia coli K-12 strains, are described. One mutant had a very low aspartate aminotransferase ( l -aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1, formerly known as glutamate-oxaloacetate transaminase) activity (10% of wild-type activity). Another mutant lacked aspartate ammonialysae ( l -aspartate ammonia-lysae, EC 4.3.1.1, formerly known as aspartase) activity completely. These two mutants were unable to grow in a glutamate-minimal medium either at 30° or at 42°. A third mutant was selected for its inability to grow on glutamate at 42° but it could utilize glutamate for growth at 30°. This mutant was shown to have a thermolabile aspartate ammonia-lyase. All three mutants exhibited normal (wild-type) levels of glutamate dehydrogenase ( l -glutamate:NADP oxido-reductase (deaminating), EC 1.4.1.4) activity. On the basis of these data and earlier findings from this and other laboratories it is concluded that the major pathway of glutamate metabolism in E. coli is via trans-amination with oxaloacetate to give α-ketoglutarate and aspartate, and subsequent deamination of the aspartate to fumarate.

5-aza-C-induced changes in the time of replication of the X chromosomes of Microtus agrestis are followed by non-random reversion to a late pattern of replication

Treatment with 5-azacytidine (5-aza-C) causes an advance in the time of replication and enhances the DNase-I sensitivity of the inactive X chromosome in Gerbillus gerbillus fibroblasts. We found that these changes were not stably inherited and upon removal of the drug the cells reverted to the original state of one active and one inactive X chromosome. In order to determine whether this reversion was random, we used a cell line of female Microtus agrestis fibroblasts in which the two X chromosomes are morphologically distinguishable. In this work we show that the reversion to a late pattern of replication is not random, and the originally late replicating X chromosome is preferentially “reinactivated”, suggesting an imprinting-like marking of one or both X chromosomes. The changes in the replication pattern of the X chromosome were associated with changes in total DNA methylation. Double treatment of cells with 5-aza-C did not alter this pattern of euchromatin activation and reinactivation. A dramatic advance in the time of replication of the entire X linked constitutive heterochromatin (XCH) region was however, observed in the doubly treated cells. This change in the replication timing of the XCH occurred in both X chromosomes and was independent of the changes observed in the euchromatic region. These observations suggest the existence of at least two independent regulatory sites which control the timing of replication of two large chromosomal regions.