Template Capacity Research Articles

Preparation and properties of chromatin from the silk glands of the silkworm Bombyx mori.

A novel method to isolate chromatin from the silk glands of the silkworm has been developed. The procedure involves digestion of the basement membrane by collagenase in the presence of the detergent Triton X-100, homogenization of the incubated glands in the presence of detergents Triton X-100 and deoxycholate and centrifugation through a linear sucrose gradient. These steps eliminate contamination by silk fibroin considerably and almost all endoplasmic reticulum membranes and ribo-somes. Further processing yields a chromatin preparation which satisfies the many criteria established by Bonner et al. (Methods Enzymol. 12 B, 3, 1968). The chromatin has the normal complement of the histone components, the relative proportion of H1 histone showing, however, an increase as the silk glands pass through the growth and secretory phases of the fifth instar. Chromatin thus isolated retains about 40% of the total RNA polymerase activity. The relative proportions of the α-amanitin-resistant (I + III) and sensitive (II) enzymes are approximately similar to that found in the whole tissue. Denatured DNA, high ionic concentrations and heparin all enhance endogenous activity. Exogenous bacterial RNA polymerase can transcribe RNA from chromatin over that of endogenous enzyme, although to a limited extent. Sonicated chromatin, particularly that portion which behaves as loose or euchromatin on sucrose gradients acts as a very good template for exogenous enzyme. Analysis of transcriptional products demonstrate the presence of ribonucleases in chromatin preparations. Comparison of endogenous enzyme activity and template capacity of chromatin from 2, 5 and 8-day-old silk glands (fifth instar) demonstrates that the decline in transcriptional activity from day 5 to day 8 is due to both a decline in enzyme activity and template capacity.

Activity of purified and chromatin-associated mouse TLT hepatoma RNA polymerases on homologous whole chromatin and euchromatin-enriched segments isolated from homologous chromatin

The RNA polymerase (EC 2.7.7.6) activity of mouse TLT hepatoma nuclei is separable into three forms, I, IIA, and IIB; each of these partially purified enzymes demonstrates characteristics generally similar to those reported for these enzymes of other systems. All three forms of TLT hepatoma RNA polymerase show a considerable preference for single-stranded DNA. The full expression of the endogenous RNA polymerase activity of mouse TLT hepatoma chromatin is dependent on the salt concentration. No additional template activity to added RNA polymerase I or II is available. Physical shearing decreased endogenous RNA polymerase activity and increased template capacity to the added enzymes. Glycerol-gradient fractionation of physically sheared chromatin gave a fairly diffuse distribution of the endogenous RNA polymerase activity to the marginally euchromatin-enriched fractions. However, enzymatic shearing of TLT hepatoma chromatin by Mg,Ca-dependent autodigestion results in a distribution of endogenous RNA polymerase activity to the highly euchromatin-enriched fractions similar to that obtained for nascent RNA (Paul, I. J. &Duerksen, J. D. (1976) Biochem. Biophys. Res. Commun. 68, 97–105). The distribution patterns of template capacity determined with added RNA polymerase I and II differed somewhat from the above distributions and varied with the length of autodigestion. Shearing by autodigestion is preferred, and followed by glycerol-gradient centrifugation permits a considerable enrichment for euchromatic segments.

Poly ADP-ribosylation of DNA-dependent RNA polymerase I from quail oviduct. Dependence on progesterone stimulation.

Progesterone causes in goblet cells of oviducts of estrogen hormone-stimulated immature quails selectively gene activation without affecting DNA synthesis. This biological model has been used to study the influence of poly ADP-ribosylation during the processes of DNA transcription. Administration of progesterone in vivo causes an increase of the activity of RNA polymerase I and II in isolated nuclei. This increase is accompanied by a marked decrease of the specific activity of poly (ADP-Rib) polymerase. After in vitro ADP-ribosylation of nuclear proteins the template capacity of chromatin for ""exogenous'' RNA synthesis (with E. coli DNA-dependent RNA polymerases) as well as for ""endogenous'' RNA synthesis with DNA dependent RNA polymerases II is not affected, whereas the data presented seem to indicate that the capacity for RNA synthesis mediated by ""endogenous'' DNA-dependent RNA polymerase I might be inhibited after ADP-ribosylation. Evidence is presented to show that a considerable amount of poly (ADP-Rib), synthesized by poly (ADP-Rib) polymerase in isolated nuclei, is linked with RNA polymerase I. The rate of synthesis of poly (ADP-Rib) is dependent on the incubation temperature (optimum at 25 degrees C) and it can be inhibited by the specific inhibitors of poly (ADP-Rib) polymerase nicotineamide, thymidine and formycin B. Poly (ADP-Rib) is probably associated with RNA polymerase I through a covalent linkage. ADP-ribosylated RNA polymerase I has been purified 550 fold with respect to the nuclear extract corresponding to a 4,000 fold purification from the whole cell homogenate. The ratio between poly (ADP-Rib), formed during preincubation of nuclei with NAD, and RNA polymerase I remains almost constant during the purification procedures. The extent of ADP-ribosylation of RNA polymerase I decreases during gene expression. Thus we conclude that poly ADP-ribosylation of this enzyme is one of the regulatory mechanisms by which specificity of DNA transcription is achieved.

Restriction of template capacity of rat liver chromatin by a non-histone peptide from calf thymus.

WE have reported1 the isolation of a thymic peptide which strongly inhibits DNA-directed RNA polymerase in vitro. We have shown that transcription is controlled at the level of initiation. The active factor was isolated from aqueous ultrafiltered thymus extracts, purified by chromatography, first on DEAE–cellulose and then on Dowex 50 WX2, and characterised as a peptide of low molecular weight (<5,000). Its biological activity cannot be attributed to the presence of a nuclease or of a histone fragment. The inhibition of RNA synthesis is due to an ionic interaction between the peptide and DNA. The physicochemical and thermodynamic properties of this interaction are described elsewhere2. We report here the effect(s) of the thymic peptide on transcription of rat liver chromatin. With some obvious limitations3, the in vitro chromatin transcription system is closer to the in vivo state than the purified DNA. Our experiments were prompted by our previous results, which demonstrated that the thymic extract modifies the physicochemical state of DNA and the interactions between DNA and nuclear proteins in the liver of old rats4,5.

Effects of estrogen and progesterone on transcription, chromatin and ovalbumin gene expression in the chick oviduct

The effects of estrogen, progesterone and estrogen + progesterone combined on nuclear transcriptional processes in oviducts of immature chicks, previously withdrawn from estrogen, are reported. The responses to the steroids of the endogenous nuclear RNA polymerase activities, both nucleolar (I) and nucleoplasmic (II), the chromatin compositions and template capacities, and the appearance of ovalbumin messenger RNA (mRNA) are compared. When immature chicks (previously treated for 14 days with estrogen) are withdrawn from estrogen treatment, there is a gradual reduction in both polymerase activities. Diurnal variations in polymerase II activities in the oviduct of withdrawn chicks required that subsequent experiments include time-matched controls. The hormones alter RNA polymerase I and II activities in vivo as assayed in isolated nuclei. Progesterone represses the polymerase I and II activities, while estrogen alone and estrogen + progesterone enhance both polymerase activities immediately after injection. Diethylstilbestrol, a synthetic estrogen, causes changes similar to those of estrogen. The effects of these steroids on the polymerases are detected within 15 min of hormone injection. Changes in the capacities of chromatins to serve as template for RNA synthesis in general correlated with changes in polymerase II activities. Interestingly, in the case of estrogen treatment, the acidic chromatin protein (but not histone) levels fluctuate positively with the template capacities of the chromatin. An antagonism between estrogen and progesterone is observed in the responses of both RNA polymerases I and II activities as well as in the chromatin template capacity. Levels of messenger RNA coding for ovalbumin, as detected by hybridization with labeled complementary DNA, increase in oviducts of withdrawn chicks within 2–3 h of the injection of estrogen, progesterone or estrogen + progesterone. This rapid accumulation of ovalbumin mRNA is not accompanied in each case by a similar increase in polymerase II activity or chromatin template capacity.

Proteins solubilized from frog virus 3 particles: effect on transcription.

The treatment of KB cells with viral proteins solubilized from frog virus 3 particles (SVE) induced a rapid shutoff of host RNA synthesis. The RNA polymerase activities of SVE-treated cells were drastically depressed, corresonding, at least for RNA polymerase B, to a decrease in the number of enzyme molecules. In vitro, SVE had no direct effect on RNA polymerases but was capable of binding with calf thymus DNA to form an SVE-DNA complex modifying the template capacity. The effect of SVE on a transcription system consisting of cell lysates would suggest that cytoplasmic factors are necessary for expression of the inhibitory capacity of SVE.

Increased intestinal chromatin template activity. Influence of 1alpha,25-dihydroxyvitamin D3 and hormone-receptor complexes.

1alpha,25-Dihydroxyvitamin D3 administration to rachitic chicks results in an increase in the chromatin template activity of intestinal target tissue assayed in vitro using Escherichia coli RNA polymerase. The maximum stimulation of template capacity was 12 to 20% over control values and occurred 2 hours after administration of the sterol. This rapid effect preceded the biologic response to 1alpha,25-dihydroxyvitamin D3 in the intestine and was not observed in other tissues such as liver or kidney. The in vivo enhancement of intestinal chromatin template activity was specific for the 1alpha,25-dihydroxyvitamin D3 hormone in that equivalent doses of 25-hydroxyvitamin D3 or vitamin D3 did not elicit a response in 2 to 3 hours. Only 1alpha-hydroxyvitamin D3, a synthetic sterol which is very rapidly metabolized to the 1alpha,25-dihydroxyvitamin D3 form, was able to minic the natural hormone in vivo. To further elucidate the nuclear mechanism of action of 1alpha,25-dihydroxyvitamin D3, the hormone was preincubated at 0 degrees with intestinal cytosol to form hormone-receptor complexes. After addition of the hormone-receptor complexes to purified intestinal mucosa nuclei and incubation for 1 hour at 25 degrees, chromatin isolated from this reconstituted system displayed a significant increase in template activity as compared to chromatin prepared from similar in vitro incubations not containing hormone. This stimulation was 12 to 24% over control values and exhibited an absolute requirement for intestinal cell cytosol. The response was specific for physiologic levels of 1alpha,25-dihydroxyvitamin D3, but occurred with pharmacologic doses of 25-hydroxyvitamin D3. It is concluded that a stimulation of the chromatin template activity of intestinal target tissue by 1alpha,25-dihydroxyvitamin D3 may be an integral part of the ultimate physiologic response of enhanced calcium transport.

Growth hormone and drug metabolism. Acute effects on nuclear ribonucleic acid polymerase activity and chromatin.

Adult male rats, subjected either to sham operation or to hypophysectomy and adrenalectomy were maintained for 10 days before treatment with growth hormone. Results of the acute effects of growth hormone on the rat liver nuclear RNA polymerase I (nucleolar) and II (nucleoplasmic) activities as well as the chromatin template capacity were then studied and compared with the growth-hormone effects on the drug metabolism described in the preceding paper (Wilson & Spelsberg, 1976). 2. Conditions for isolation and storage of nuclei for maintenance of optimal polymerase activities are described. It is verified that the assays for polymerase activities require a DNA template, all four nucleoside triphosphates, and a bivalent cation, and that the acid-insoluble radioactive product represents RNA. Proof is presented that under high-salt conditions DNA-like RNA (polymerase II) is synthesized, and that under low-salt conditions in the presence of alpha-amanitin, rRNA (polymerase I) is synthesized. 3. In the livers of hypophysectomized/adrenalectomized rats, growth hormone increases the activity of both RNA polymerase enzymes and the chromatin template capacity within 1h after treatment. The effects last for 12h in the case of polymerase II but for only 6h in the case of polymerase I. Sham-operated rats respond to growth hormone in a manner somewhat similar to that shown by hypophysectomized/adrenalectomized rats. These results, which demonstrate an enhancement of RNA polymerase I activity in response to growth hormone, support those from other laboratories. 4. Growth-hormone enhancement of the chromatin template capacity in the liver of hypophysectomized/adrenalectomized rats contrasts with previous reports. The growth-hormone-induced de-repression of the chromatin DNA could represent the basis of the growth-hormone-induced enhancement of RNA polymerase II activity in the hypophysectomized/adrenalectomized rats, although some effect of growth-hormone on the polymerase enzymes is still suggested.

Early androgen action in kidney of normal and androgeninsensitive ( [formula omitted]) mice Changes in RNA polymerase and chromatin template activities

Intranuclear accumulation of testosterone was compared with early changes in transcriptional events in kidneys from normal female and androgen-insensitive ( tfm y ) mice. Following a subcutaneous injection of [ 3H] testosterone, total nuclear uptake of the steroid was maximal at 30 min and declined to about 40% of the peak value by 4 h after hormone administration. After a single subcutaneous dose of testosterone, RNA polymerase activity assayed in intact nuclei in the presence of Mg 2+ and α-amanitin (nucleolar RNA polymerase I), as well as the enzyme activity sensitive to low concentration of the toxin (nucleoplasmic RNA polymerase II), increased within 15 min and attained peak values at 2 and 1 h, respectively. The activity of both polymerases declined almost to the control level by 4 h and then increased again with a second peak at 20 and 12 h for RNA polymerase I and II, respectively. Similarly, the template capacity of mouse kidney chromatin, as measured with mammalian RNA polymerase II, increased by 15 min, reached a peak at 1 h and returned to control level by 4 h following hormone treatment. A second dose of testosterone given at the nadir (4 h) was not capable of stimulating renal chromatin template activity significantly as compared to the effect observed after the initial hormone treatment. Contrary to the testosterone-stimulated changes in transcriptional events observed in normal female mice, androgens elicited no response in androgen-insensitive tfm y mice, animals lacking cytosol androgen receptors. These results strongly support the contention that hormone-specific receptors are obligatory to steroid-mediated modifications in gene transcription.

Non-histone chromosomal proteins. Their isolation and role in determining specificity of transcription in vitro.

We describe a method for fractionation of chromatin components by selective dissociation with salt in buffers containing 5 M urea in combination with cromatography on hydroxyapatite at 4 degrees C. This results in two histone and four non-histone fractions which are recovered in high yield and with minimal proteolytic contamination. Template capacity measurements of the isolated chromatins and pre-saturation competition hybridization experiments support the idea that a group of non-histone proteins activate the transcription of specific DNA sequences which were not transcribed from purified DNA to the same extent. In reconstitution experiments a non-histone protein fraction, NH4, prepared from lymphocyte chromatin by hydroxyapatite chromatography is shown to cause transcription in vitro of lymphocyte-specific RNA sequences. A subfraction with a molecular weight of 30 000 comprising 40% of the NH4 fraction protein is characteristic for this tissue and not found in liver chromatin.

The interaction of natural tetra-azacyclopentazulene dyes with DNA and their effects on the DNA AND RNA polymerase reactions

The interaction of two natural tetra-azacyclopentazulene dyes with native calf thymus DNA was studied by means of microcalorimetric, viscosimetric, and spectroscopic measurements. The results are consistent with the hypothesis of an intercalative-binding. However, comparison of calorimetric studies shows that the changes in enthalpy associated with the interaction of these compounds with DNA are, in absolute value, significantly lower than those found with known intercalating agnets (daunomycin, ethidium bromide). The influence of these dyes on the template capacity of DNA in the in vitro synthesis of nucleic acids was also determined. Under the conditions used, these compounds selectively inhibited DNA synthesis. No appreciable inhibitory effect upon E. coli RNA polymerase was observed. Both compounds had greater inhibitory effect on rat liver high molecular weight DNA polymerase than E. coli DNA polymerase I. Zoanthoxanthin was a more effective inhibitor than 3-norzoanthoxanthin.

Transcription in vitro of DNA in isolated bacterial nucleoids

The in vitro transcription of DNA in isolated nucleoids from Escherichia coli was investigated. The purpose of the study was to determine the effects on DNA transcription of using as template a condensed bacterial chromosome. To compare the template capacity of the DNA in the condensed chromosome with that of the completely unfolded chromosome, very high RNA polymerase/DNA ratios were used during transcription so that the amount of DNA was limiting. The rate of total RNA synthesis was greater on the condensed chromosome. The RNA chain elongation rate and the average size of the RNA transcripts were similar on the condensed and unfolded templates; however, RNA chain initiation rates were greater on the condensed chromosome. The results show that the maximum number of RNA polymerase molecules which can simultaneously synthesize RNA is greater on the condensed chromosome. A preferential synthesis of ribosomal RNA was observed on both the condensed and unfolded chromosomes. Although the absolute rate of rRNA synthesis was greater on the condensed template, the rate of rRNA synthesis relative to the rate of total RNA synthesis was similar on both templates. The results suggest that the genes specifying rRNA do not differ significantly in their availability for transcription relative to the remainder of the genome in folded and unfolded chromosomes. Moderate concentrations of spermidine stabilized the condensed state of the folded chromosome during transcription and the average size of the isolated nucleoids, as estimated from sedimentation studies and direct microscopic visualization, remained unchanged during transcription.

Nuclear receptors for thyroid hormone: evidence for nonrandom distribution within chromatin.

Chromatin receptor proteins appear to mediate some actions of thyroid hormone. In this study, sheared mammalian chromatin containing [125I]triiodothyronine (T3) bound by these receptors was separated using sucrose gradient velocity sedimentation. T3-receptor complexes were distributed throughout the chromatin fractions, but were enriched in the slowly sedimenting fractions. The latter contain most of the template capacity for RNA synthesis and most of the endogenous RNA polymerase activity but a minor portion of the total DNA. Formaldehyde treatment of chromatin containing receptor-bound [125 I ]T3 resulted in fixation of radioactivity, as evidenced by its migration with chromatin after equilibrium density gradient sedimentation in both cesium chloride and Conray. This fixation implies that the T3 receptor protein is closely associated with chromatin. These results suggest that proteins involved in the regulation of gene function may be nonrandomly distributed within chromatin subfractions, and are consistent with a direct role for thyroid hormone in regulating genetic expression.

Modification of the template capacity of liver chromatin for form-B ribonucleic acid polymerase by food intake in rats under controlled feeding schedules.

Nuclei from liver of rats accustomed to eating during the first 8h of a daily 12h dark period demonstrate an increased capacity to synthesize RNA 6H after the beginning of the feeding period. 2. This increase is accompanied by a higher yield of extractable form-B DNA-dependent RNA polymerase activity. 3. The endogenous RNA polymerase activity associated with nuclear chromatin is also stimulated by food intake. Both purified and chromatin-associated form-B enzyme activities exhibit different ionic strength requirements after food intake. 4. The sensitivity of exogenous (added) form-B-enzyme to changes in ionic strength changes after feeding when chromatin is used as template. 5. Chromatin extracted from the liver of fed rats is a better template for form-B-enzyme than chromatin extracted from starved rats.

MECHANISM OF ACTION OF FEMALE SEX STEROIDS

Literature on the mechanism of action of female sex steroids is reviewed. Generally the steroid enters the target cell by diffusion through the plasma membrane (this may be energy dependent or protein mediated) combines in the cytoplasm with a specific steroid receptor protein and is then activated by a temperature and salt-dependent process which forms a transformed steroid-receptor complex. This activated complex is then translocated possibly by a simple diffusion to the nucleus where nuclear receptor complex binds to acceptor protein on the chromatin. This facilitates the synthesis of different RNAs by activating RNA polymerases and increasing chromatin template capacity. These newly formed precursors of mRNA rRNA and tRNA are processed and transported to the cytoplasm. Steroid specific mRNAs are then translated on the polyribosomes resulting in the formation of hormone specific proteins and other cellular components required for the change in target cell function. These phases in the cellular action of estrogens and progesterone are discussed in detail.

Studies on the RNA synthesized in the ovary of immature rats after HCG administration (author's transl)

Although many researchers have reported that RNA synthesis in the ovary is enhanced by gonadotropin treatment, there are only a few papers concerning the character of newly synthesized RNA after gonadotropin treatment. In this paper, the RNA synthesized in the ovary of immature rats after HCG treatment was qualitatively studied. Immature female Sprague-Dawley rats were administered with 0.3 mc per rat of 3H-uridine at a certain time interval after injection of HCG (10 iu/rat) and the ovaries were subsequently isolated after 15, 30 or 60 minutes. RNA was extracted from the homogenate of the ovaries according to the hot phenol method after Scherrer and Darnell. The 3H-RNA thus extracted was treated with electrophoretically purified DNase to break down and remove DNA that mingled with it. The RNA solution ultimately obtained was analysed on a 3-20% sucrose gradient. The different fractions thus separated were then subjected to measurement of radioactivity and optical density at 260 mmug. The RNA extracted from the ovary of immature untreated rat labeled with 3H-uridine for 15 minutes showed a flat pattern of radioactivity from the top to the bottom fractions with low radioactivity. Otherwise, when labeled for one hour, the RNA showed a pattern of radioactivity like those of optical density at 260 mumu with peaks of r-RNAs and t-RNA. When the ovary was pulse-labeled with 3H-uridine for 15 minutes starting 2 hours after injection of HCG, the RNA with a large S value was synthesized and the pattern of variation in radioactivity was that of rising near the bottom fraction and declining with access to the top fraction. The results obtained by labeling for 15 minutes starting 40 hours after PMS administration were similar to those obtained in immature untreated rats. The patterns of radioactivity in RNA obtained by the labeling for 15 minutes starting 2 hours after HCG and 42 hours after PMS were similar to those starting 2 hours after only HCG injection. The patterns of radioactivity became similar to those of optical density at 260 mmu, when the ovaries were labeled for 30 or 60 minutes. From these results, it was suggested that the newly synthesized RNA 2 hours after HCG was constructed from m-RMA with rapid turn over and precursors of r-RNAs and t-RNA. This RNA synthesis was blocked by pretreatment with actinomycin but not by cycloheximide. From these results, it was suggested that enhancement in RNA polymerase activity or change in template capacity of DNA which would have an effect on RNA synthesis was not based on newly synthesized protein.

Template capacity of mouse TLT hepatoma ghromatin fractions

1. 1. Isolated mouse TLT hepatoma chromatin has no measurable protease activity, whereas some of the chromatin fragments separated on exclusion agarose columns possess endogenous RNA polymerase activity. 2. 2. The template capacity of the template active chromatin fractions is 5–50-fold greater than usual when measured using a portion of E. coli RNA polymerase, apparently modified by purification and aging. 3. 3. Any contaminating nuclease activity in partially-purified RNA polymerase had minimal modifying effects on template capacity; the addition of pancreatic DNase but not RNase essentially eliminates the template capacity of chromatin fractions. 4. 4. Under conditions avoiding chromatin aggregation, similar template capacity patterns of chromatin fractions are obtained when the RNA incorporation reaction is executed either prior to, or subsequent to, the fractionation of the sheared chromatin.

Changes at transcriptional level in the rat liver following whole-body X-irradiation

Exposure of animals to a single lethal whole-body dose of X-rays is known to cause enhanced rates of protein synthesis in the liver during 12 to 24 hr post-irradiation. Evidence from various lines indicates that these changes are solely the consequence of a general stimulation in RNA synthesis [ 1,2]. Previous studies of ours on the mechanism by which radiation exerts stimulatory effect on RNA biosynthesis in the rat liver have revealed that it is the template capacity of chromatin rather than the level or the activity of RNA polymerase which exhibits distinct rise in the interval between 4 and 24 hr post-irradiation [3]. Several workers have recently reported that under conditions which lead to gene activation specific enhancement in synthesis of certain chromosomal acidic proteins can be discerned in animal cells. Thus in rats, selective stimulation in the synthesis of chromosomal acidic proteins has been observed in the liver following administration of cortisol [4] and glucagon [.5] and in the liver and the uterus in response to estradiol [6]. In this communication, we present evidence to show that concurrent with amplification in template capacity of liver chromatin, incorporation of labelled amino acid precursors into specific chromosomal acidic proteins in the liver is also selectively stimulated during 4 to 24 hr following whole-body exposure of rats to 1000 R X-rays.

Distribution of transcribable DNA sequences in mouse liver hepatoma chromatin.

Shearing in either a Virtis small or large volume capacity cup under defined conditions gave chromatin fragment size distribution upon agarose exclusion column chromatography (50 × 106) that varied inversely with shear time. The large- and small-sized chromatin fragments possessed high template capacity while the intermediate fragments (the majority) had relatively low template capacity; the differences in template capacity of these size groups varying inversely with the shear time. The protein/DNA and RNA/DNA ratios remained relatively constant in all size groups.

Globin messenger RNA synthesis and processing during haemoglobin induction in Friend cells. II. Evidence for post-transcriptional control in clone 707

Clone 707 of the Friend cell, from which clone M2 was derived, also responds to treatment with dimethylsulphoxide by increased synthesis of haemoglobin. However, although the final globin mRNA content of fully induced cells is comparable, the basal level is considerably higher. Moreover, there is no increase in the number of globin mRNA molecules in the nucleus as a result of induction and there is little, if any, difference in the template capacity for globin gene transcription of chromatin from induced and uninduced cells. It is postulated that clone M2 represents the original line, exhibiting linked transcriptional and post-transcriptional control of haemoglobin synthesis and that clone 707 now consists mainly of a variant in which transcriptional control is relaxed.