Transcriptional frequency and cell determination.

The relative base composition of DNA regulatory sequences of certain genes of undetermined multipotent progenitor cells may account for the frequency of transcription of these genes in cell determination. The sequences of these regulatory regions of cell determination genes that are more AT-rich would create the potential for transcription at a higher frequency due to their lower melting temperature, as well as propensity to bend. An increase of one or more of the high mobility group (HMG) chromatin proteins would preferentially bind the more AT-rich regulatory sequences, thereby increasing the rate of transcription. The amount of unphosphorylated H1 histone reacting with these same regulatory sites may decrease transcription frequency. The level of cell growth, i.e. total protein synthesis of a cell, is correlated positively with the synthesis of HMG proteins. H1 histone synthesis is linked to DNA replication. Unbalanced growth would alter the amounts of HMG proteins and H1 histone, thus changing transcriptional frequency. The greater the enrichment of AT sequences in the regulatory regions of the cell determination genes, the greater may be the extent of evolutionary conservation. Higher frequency of transcription of the cell determination genes with the more AT-rich regulatory sequences could account for the earlier expression of the more conserved cell determination genes during embryonic development. Preferential binding of H1 histone to the more AT-rich regulatory sequences would subsequently restrict their transcription before that of less conserved cell determination genes.

Cell proliferation and protein synthesis as initial factors in determination of axial polarity.

The rate of cell proliferation relative to that of protein synthesis appears to have an initial role in establishment of axial polarities in developing animal embryos. An increase in this ratio leads to anterior or dorsal differentiation, while reduction allows posterior or ventral differentiation in a number of organisms. The role that various growth factors play in the regulation of proliferation and protein synthesis is examined.

Induction of tumor cell differentiation.

It is proposed that cell proliferation with reduced individual cell growth (total protein accumulation) is necessary, but not sufficient, for cell differentiation. These conditions may facilitate transcription and accumulation of histones H1 and/or H1o relative to the core histones. This may have a critical role in cell differentiation.

Hierarchical differentiation of multipotent progenitor cells.

Selection of a pathway of differentiation in multipotent progenitor cells may depend on the amount of histone H1 or H1 zero relative to the core histones. With low levels of these linker histones, it is proposed that an evolutionarily more ancient cell differentiation occurs. Greater repetition of AT-rich regulatory motifs allows more frequent, hence earlier, transcription of genes, accounting for this type of cell differentiation. It is further proposed that a decrease of total cell protein accumulation leads to an increase of histone H1 or H1 zero relative to the core histones. It is suggested that these linker histones preferentially bind the more AT-rich regulatory sequences, thereby restricting the phylogenetically more ancient differentiation potency. This allows differentiation of an evolutionarily younger cell type.

Histone H1o ribosomal RNA synthesis and cell differentiation.

It is proposed that histone H1o prevents binding of the rRNA transcription factor UBF to regulatory sequences of the rRNA gene. This has a role in reducing rRNA synthesis and inhibiting cell proliferation. This affords the opportunity for other mechanisms to promote cell differentiation.

Possible role of H1 histone in the differentiation of mouse erythroleukemia cells.

Serum-starved mouse erythroleukemia cells, stationary phase cells or cells cultured in dibutyryl cAMP (1 mM) can be induced to differentiate by addition of 20% fetal calf serum plus cycloheximide. Culturing unstarved log phase cells in 20% fetal calf serum plus low levels of cycloheximide and histone H1 also causes a significant level of differentiation. These same concentrations of cycloheximide and H1 histone employed separately with 20% fetal calf serum do not induce differentiation. The role these procedures may have in causing an accumulation of histone H1 and cell differentiation is discussed.

Cell growth and division in relation to differentiation of mouse erythroleukemia cells.

Addition of certain inhibitors of RNA or protein synthesis to cells cultured in 20% serum, but not 5% serum, induces the differentiation of mouse erythroleukemia cells. Differentiation also was induced by culture at a low serum concentration (0.1%) to starve cells to quiescence, then inducing division by exposing cells to either of two high-pH regimes.

Protein kinase activity, growth and differentiation of murine erythroleukemia cells.

The addition of type 2 cAMP-dependent protein kinase stimulates division of cultured Friend erythroleukemia cells. A synthetic peptide representing the inhibitory portion of the heat stable protein kinase inhibitor protein and an inhibitor of cAMP-dependent protein kinase (N-(2-aminoethyl)-5-isoquinolinesulfonamide dihydrochloride or H-9) inhibited cell division. The latter inhibitor (H-9) induced differentiation of the cells.

Factors controlling the size of DNA loops in frog embryos and Friend erythroleukemia cells.

Measurements were made of the average size of fluorescent halos of nucleoids of developing frog embryos, mouse fibroblasts (A9 cells) and Friend erythroleukemia cells cultured with various compounds. These halos are thought to represent relaxed lengths of loops of DNA attached to the nuclear matrix. Measurement of transcription of RNA from nuclei in vitro suggested that cells with more loops (smaller halos) had more initiation sites for transcription. DNA loop number decreases during development of the frog embryos, and red blood cells of adults have even fewer and larger loops. One or two days of culture of Friend cells in various compounds that induce differentiation resulted in more DNA loops than for control cells, but after 5 days of culture the differentiated erythrocytes had fewer DNA loops than control cells at stationary phase. Stationary phase cells have almost twice as many DNA loops as log phase cells. Various compounds that stall cells at the G1/S border induced more DNA loops, but subsequently the number of DNA loops decreased as the cells entered S phase. A number of compounds which slow the rate of cell division cause the formation of more DNA loops, perhaps due to longer G1 periods.

Localization of AT-rich sequences at the nuclear matrix.

After culture with 3H-thymidine murine erythroleukemia cells with more sites of DNA attached to the nuclear matrix have more labeled thymidine in their matrix-bound DNA than in their total DNA than do cells with fewer attachment sites. This indicates that the average attachment site sequence is enriched in A + T base pairs.

Effect of polycations and polyanions on cell division of cultured mammalian cells.

The polycations (H1 histone and polylysine) and polyanions (heparin and various RNA preparations) stimulate cell division of cultured mammalian cells. The mechanisms by which both polycations (H1 histone and polylysine) and polyanions (heparin and RNA) may increase the rate of cell division are discussed.

The effect of induction of hemoglobin synthesis in cultured Friend cells on the number of initiation sites for replication and transcription.

It was found that differentiation of murine erythroleukemia cells can be induced by 5-fluorodeoxyuridine (FudR), amethopterin and alpha-aminoisobutyrate. Each of these compounds is believed to delay the onset of DNA synthesis. Since relief of the FudR block to DNA synthesis by addition of thymidine can increase the number of initiation sites for replication (Taylor, 1977), the effect of various inducers and inhibitors of differentiation of Friend cells upon the relative number of initiation sites for replication and transcription was investigated. Very efficient inducers of hemoglobin synthesis, hexamethylene bisacetamide (HMBA) and dimethylsulfoxide (DMSO), increase the number of initiation sites for transcription and HMBA also increases the number of functional initiation sites for replication. Two other compounds that induce differentiation of Friend cells, low levels of actinomycin D and FudR, did not increase the number of initiation sites for transcription. Compounds that prevent induction of hemoglobin synthesis by HMBA and DMSO include 5-bromodeoxyuridine (BrdU) and novobiocin. Both of these compounds were found to decrease the number of functional initiation sites for transcription and it is known that both compounds reduce the number of initiation sites for replication. The relation between initiation of replication and transcription, and its effect upon differentiation of erythroleukemia cells is discussed.

Relative base composition of initiation sites for transcription in developing frog embryos.

The use of an inhibitor (0.4 N (NH4)2SO4) and an activator (sodium polydextran sulfate) of initiation of transcription in nuclei of frog embryos in vitro indicated that initiation sites for transcription are relatively A--T-rich. Comparison of incorporation of labeled ATP, UTP, and GTP, when initiation of transcription is prevented, indicates that initiation sites become relatively less A--T-rich as development proceeds. Inhibition of initiation of transcription in isolated nuclei blocks the normal increase in rate of transcription that occurs when cells enter the S phase, showing that an increased level of transcription depends upon a greater number of initiation sites.

The effect of added H1 histone and polylysine on DNA synthesis and cell division of cultured mammalian cells.

Addition of H1 histone or polylysine (10 microgram/ml) to cultured Friend erythroleukemia cells or to two mouse lymphoma cell lines (el-4 and S-49) increased levels of cell division in these cultures. There is a stimulation of incorporation of labeled thymidine into DNA in cultures containing H1 histone and polylysine. DNA fiber autoradiographic experiments revealed that replicon size is decreased in the cells cultured with H1 histone and polylysine at later periods of culture.

Replicon size increase due to 5-bromodeoxyuridine incorporation.

The incorporation of 5-bromodeoxyuridine into DNA of cultured minnow and hepatoma cells results in the formation of larger replicons.