Nuclease sensitivity of alpha-fetoprotein, metallothionein-1, and immunoglobulin gene sequences in mouse during development.

The production of alpha-fetoprotein (AFP) and metallothionein-1 (MT-1) in mouse tissues follows a well-defined developmental pattern. The genes for these proteins are highly transcribed in embryo liver but transcribed at a very low rate in adult liver and in brain at all stages of development. A dot hybridization procedure was defined for quantitative screening for AFP, MT-1, immunoglobulin, and satellite DNA sequences to determine the relative degree of micrococcal nuclease sensitivity of these DNA sequences in fetal, newborn, and adult liver and brain, and the visceral yolk sac of the embryo. It was found that, for the DNA sequences assayed, three distinct chromatin conformations exist. DNA that does not code for protein (satellite DNA) was highly resistant to nuclease cleavage. DNA that codes for protein, but is not available for transcription (unrearranged immunoglobulin (C mu) genes in brain, liver, and yolk sac) was fourfold more sensitive to cleavage than were satellite DNA sequences. A further sevenfold increase in nuclease sensitivity was detected in genes actively being transcribed (MT-1 and AFP genes in embryo liver). Quiescent MT-1 and AFP genes were intermediate in nuclease-sensitivity between active genes and unrearranged C mu genes. These data indicate that MT-1 and AFP genes are permanently established in a nuclease-sensitive chromatin conformation early in liver development, and that conformation is maintained regardless of the degree of transcription of the genes. A second, reversible change in chromatin structure occurs in step with changes in the degree of developmentally regulated expression of AFP and MT-1 genes.

Mouse hepatic metallothionein-I gene cleavage by micrococcal nuclease is enhanced after induction by cadmium.

Micrococcal nuclease has been shown to preferentially cleave chromatin in the region of genes actively engaged in transcription. We have used this preferential cleavage to show that the metallothionein (MT) gene in adult mouse liver, when induced to produce mRNA by injection of cadmium, becomes more susceptible to nuclease cleavage. However, the MT gene in uninduced liver, and the alphafoetal protein (AFP) gene in both induced and uninduced liver, remain relatively resistant to nuclease cleavage. The AFP gene is not normally expressed in cadmium induced or uninduced liver. Thus, susceptibility of genes to nuclease cleavage appears to rise with increasing transcription of the gene.

Further characterization of isolated presumptive euchromatin fractions of mouse hepatoma cells utilizing a cDNA probe complementary to the homologous poly(A)+ mRNA.

Poly(A)+ mRNA from mouse hepatoma ascites cell cytoplasm is characterized by three frequency classes: an abundant frequency class of a limited number of different nucleotide sequences, a less abundant frequency class of a larger number of different nucleotide sequences, and a rare frequency class containing a high number of different nucleotide sequences. [3H]cDNA synthesized on this poly(A)+ mRNA template hybridizes with some of the DNAs of the putative transcribable euchromatin fraction at a significantly faster rate than with total DNA if residual contaminating RNA is not removed. Following NaOH incubation to remove such RNA, the cDNA probe hybridized with essentially the same rate to the euchromatin fractions and total DNA. Nick translation of the nuclease-sensitive sequences of chromatin demonstrated that, even with limited nuclease digestion, the excised sequences rapidly converted to small oligonucleotides. The nick-translatable, small chromatin segments showed no enrichment for transcribable sequences. Chromatin segments, which distribute to the 50S-70S glycerol gradient fractions and which satisfy several of the presumptive criteria for enrichment for transcribable sequences, therefore show no enrichment for sequences complementary to the cDNA for poly(A)+ mRNA.

Periodicity and fragment size of DNA from mouse TLT hepatoma chromatin and chromatin fractions using endogenous and exogenous nucleases.

The action of micrococcal nuclease, DNase I and DNase II on mouse TLT hepatoma chromatin revealing the periodicity of its structure as visualized by denaturing and non-denaturing gel electrophoresis, was consistent with the action of these enzymes on other chromatins. Micrococcal nuclease showed a complex subnucleosome fragment pattern based on multiples of 10 base pairs with a prominant couplet at 140/160 base pairs and the absence of the 80 base pair fragment. This couplet of the core and minimal nucleosome fragments was conspicuously present in the mononucleosomes found in the 11S fractions of a glycerol gradient centrifugation. DNase I and II produced a fairly even distribution of a 10 base pair increasing series of fragments to about 180 base pairs, a pattern also repeated in the DNA of nucleosome glycerol-gradient fractions. In limited digestions by these nucleases multinucleosomic DNA fragments are pronounced. These fragment lengths are multiples of an estimated average repeat length of nucleosome DNA of 180 base pairs. The action of the endogenous Mg/Ca-stimulated endonuclease produced only limited cuts in the hepatoma chromatin resulting primarily in multi-nucleosomic DNA fragment lengths and only upon lengthy digestion limited subnucleosomic, 10-base-pair multiple fragments are produced. The putative euchromatin-enriched fractions (50-75S) of the glycerol gradient centrifugation of autodigested chromatin, similarly, contained primarily the multinucleosomic DNA fragment lengths. These results are consistent with our previous electron microscopic demonstration that autodigested chromatin as well as the putative euchromatin-enriched fractions were composed of multi-nucleosomic chromatin segments containing a full complement of histones.

Analyses of relative protein content and distribution of histones in euchromatic and heterochromatic fractions.

Euchromatic and heterochromatic fractions obtained by autodigestion of mouse TLT (taper liver tumour) hepatoma chromatin (Paul, I. J & Duerksen, J. D. (1976) Arch. Biochem. Biophys. 174, 491-505) were analyzed for relative protein content and histone content. With one exception, all fractions had the same DNA to protein ratio. Similarly, the total histone to DNA ratio was also constant in all fractions. In addition, the relative contents of the major histones, H1, (H2A + H2B + H3), and H4, were also constant in all fractions.

Satellite DNA sequence content of polylysine-titratable and nuclease-resistant fractions of mouse liver hepatoma chromatin.

Micrococcal nuclease digestion of mouse TLT liver hepatoma chromatin proceeds rapidly to the point where approximately 35% of the DNA is recoverable by centrifugation of the chromatin DNA through 3M CsCl. The satellite DNA sequence content of this recoverable DNA is the same as whole chromatin DNA (10%). The 11s (penultimate digestion product) monomer, as well as intermediate multiples and relatively undigested large chromatin segments are separable on steep hlycerol gradients. The DNA isolated from these fractions also contains the normal 10% satellite DNA content. Progressive polylysine titration of chromatin followed by nuclease digestion gives anomalous recoveries of DNA but, nonetheless, the satellite sequence content titration of chromatin, followed by pronase and then nuclease digestion, again gave recoverable DNA with a satellite sequence content of 10%. These results are discussed in terms of the conclusion that nucleosome (or upsilon-body) structures are distributed in a random fashion over the genome.

Chromatin-associated RNA content of heterochromatin and euchromatin.

Chromatin from TLT hepatoma cells, mouse liver cells, and mouse brain cells was fractionated by differential centrifugation into a pellet, enriched with heterochromatin, and a supernatant, enriched with euchromatin. The pellet was found to contain more than twice as much of a particular species of chromatin-associated RNA per milligram chromatin DNA as did the supernatant. This chromatin-associated RNA was also found to be associated with the transcriptionally inactive chromatin of mature avian erythrocytes. Bull sperm, whose genome is known to be completely inactive, was used as the source in the preparation of sperm heads. Bull sperm head RNA appeared to consist of a single, low molecular weight species which migrated on polyacrylamide gels at a rate just slightly slower than the aforementioned chromatin-associated RNA. The results are interpreted as indicating that this chromatin-associated RNA is more prevalently associated with the heterochromatic fraction of chromatin. It is postulated that this chromatin-associated RNA might constitute a structural component of heterochromatin.

Comparison of the catalytic and immunological properties of beta-glucosidases from three strains of Saccharomyces lactis.

beta-Glucosidase from Saccharomyces lactis strains Y-123 (B(h)), Y-14 (B(m)), and Y-1057A (B(1)) was partially purified. The pH optima, Michaelis constants, and activation energies were determined for the hydrolysis of p-nitro-phenyl-beta-d-glucoside by each of the enzymes. Differences among these constants were not enough to account for the low specific activity of beta-glucosidase in strains Y-14 and Y-1057A. Enzyme-inhibitor constants were measured for a series of alkyl and aryl glucosides. In general, the three enzymes are arylglucosidases. Tris(hydroxymethyl)-aminomethane inhibited all three enzymes in an uncompetitive fashion. The inhibition was antagonized by Mg(++). An antiserum was prepared to the highly purified (200-fold) beta-glucosidase from strain Y-123. The nature and degree of cross-reaction between the three beta-glucosidases was investigated by double diffusion in agar and neutralization tests. Spur formation in the immunodiffusion tests and similar equivalence points in the neutralization tests indicated a strong degree of cross-reaction between the three enzymes. The ratio of enzyme activity to antigenicity was used to compare the relative molecular activity of beta-glucosidase in the three strains. Each strain produced the same amount of beta-glucosidase per milligram of cell protein. The results are consistent either with a lower turnover number for the beta-glucosidase in strains Y-14 and Y-1057A or with the production of beta-glucosidase with a "normal" turnover number and enough cross-reacting material to effectively reduce the specific activity to the observed levels.

Purification of beta-glucosidase from Saccharomyces lactis strain Y-123.

Saccharomyces lactis strain Y-123, a constitutive high producer of beta-glucosidase (B(h)), was grown in an enriched medium. beta-Glucosidase, extracted most easily by cell autolysis, was purified by successive ammonium sulfate precipitation, ethyl alcohol precipitation, gel filtration, calcium phosphate gel adsorption-elution, and hydroxyapatite column chromatography. The specific activity of the enzyme increased 200-fold during the purification. The electrophoretic and catalytic properties of the enzyme did not change during the procedure. Polyacrylamide gel disc electrophoresis of the partially purified enzyme revealed one major and several minor protein-staining bands. beta-Glucosidase activity in the polyacrylamide gel columns was measured directly by assaying sections of columns frozen and sliced immediately after electrophoresis. Most of the activity coincided with the major protein-staining band. Prolonged assay produced minor activity coinciding with the less intense protein bands. Properties of the enzyme suggest that it is a single, unconjugated, intracellular, high molecular weight protein. The purification procedure is applicable to strains of S. lactis which possess alleles of the B locus for beta-glucosidase synthesis.

Purification of beta-glucosidase from Saccharomyces lactis strains Y-14 and Y-1057A.

Two strains of Saccharomyces lactis (Y-14 and Y-1057A), medium (B(m)) and low (B(1)) constitutive producers of beta-glucosidase, were grown in enriched medium. beta-Glucosidase was extracted by autolysis and purified by ammonium sulfate precipitation, gel filtration, and calcium phosphate gel adsorption-elution. The kinetics of release, purification, and stability of beta-glucosidase from strains Y-14 and Y-1057A were compared with the enzyme from strain Y-123. The ability of glycerol, sorbitol, and mannitol to stabilize the beta-glucosidases is presented. A lower molecular weight, labile form of the Y-14 enzyme is demonstrated. Differences in the initial specific activites of beta-glucosidase among the three strains are discussed.

Purification and characterization of yeast beta-glucosidases.

Constitutive beta-glucosidases from Saccharomyces fragilis (Y-18) and S. dobzhanskii (Y-19) precipitated at the same concentration of ammonium sulfate. The partially purified enzymes had similar activation energies, molecular weights, affinities for certain natural and synthetic beta-glucosides, and optimal pH values for substrate hydrolysis, and they were stable over approximately the same pH range. The enzymes, however, could be clearly distinguished by other criteria. Affinities of the synthetic, sulfur-containing beta-glucosides for Y-18 enzyme were many times greater than for Y-19 enzyme. The latter enzyme was more resistant to heat. The two enzymes eluted from diethylaminoethyl cellulose at different concentrations of sodium chloride. In precipitin tests, homologous enzyme-antisera systems were highly specific. The beta-glucosidase synthesized by a hybrid, S. fragilis x S. dobzhanskii (Y-42), was unique. Characterization of this enzyme produced values which were intermediate to those for the enzymes from the parental yeast strains. Heat-inactivation slopes and Lineweaver-Burk plots for the Y-42 enzyme were anomalous. It is suggested that hydrolytic activity in Y-42 preparations is due to a spectrum of hybrid enzyme molecules composed of varying amounts of two distinct polypeptides. It is further suggested that these polypeptides may be identical to those synthesized by the parental Y-18 and Y-19 yeast strains.

Evidence for multiple molecular forms of yeast beta-glucosidase in a hybrid yeast.

A mixture of beta-glucosidases from Saccharomyces fragilis (Y-18) and S. dobzhanskii (Y-19) eluted from diethylaminoethyl cellulose in two peaks, whereas the enzyme from a hybrid, S. fragilis x S. dobzhanskii (Y-42), eluted in a single broad peak. The highest Y-42 activity fractions eluted at a sodium chloride molarity which was intermediate to the molarities at which most of the Y-18 and Y-19 activity was eluted. In cellulose polyacetate strips, Y-42 enzyme migrated as a diffuse band which spanned the distances migrated by the enzymes from the parent yeast strains. Antisera against either Y-18 or Y-19 enzyme precipitated 80 to 90% of Y-42 enzyme activity. When Y-42 enzyme was dissociated by heat or urea and reacted with parental antiserum, a concomitant increase in the opposite parental activity was demonstrable in both precipitation and complement-fixation (CF) tests. Urea-dissociated beta-glucosidases were resolvable by sucrose-gradient centrifugation into multiple bands displaying specific CF activity. When the enzymes were exposed to 4 m urea for 12 min, particles of approximately 110,000 molecular weight were obtained. By extending the exposure time to 40 min, and incorporating 0.5 m urea in the gradients, smaller particles were detected with molecular weights ranging from 18,000 to 23,000. Attempts to regenerate enzyme activity after dissociation with urea were only moderately successful. Results suggested that a slightly acidic environment favored reassociation, as did the presence of 2-mercaptoethanol. Residual urea also seemed important. It is proposed that the structural genes for both Y-18 and Y-19 enzyme are present in Y-42 cells with either independent or closely interacting regulatory mechanisms. Since synthesis of the two parental-type polypeptides may be unequal, the availability of enzyme subunits for subsequent polymerization in the cell cytoplasm might be equalized at the polysome level. Random association of subunits would produce a binomial distribution of true hybrid enzyme molecules.