STUDIES ON NUCLEAR STRUCTURE AND FUNCTION IN TETRAHYMENA PYRIFORMIS : I. RNA Synthesis in Macro- and Micronuclei

Tetrahymena in the log phase of growth were pulse labeled with uridine-³H, fixed in acetic-alcohol, extracted with DNase, and embedded in Epon. 0.5-µ sections were cut, coated with Kodak NTB-2 emulsion, and developed after suitable exposures. Grains were counted above macronuclei, above 1000 micronuclei, and above 1000 micronucleus-sized "blanks" which were situated next to micronuclei in the visual field by means of a camera lucida. An analysis of grain counts showed that micronuclei were less than ½₀₀₀ as active as macronuclei on the basis of grains per nucleus. Since micronuclei contained, on the average, about ½₀ as much DNA as macronuclei, micronuclear DNA had less than 1% of the specific activity of macronuclear DNA in RNA synthesis. However, even this small amount of apparent incorporation was not significantly different from zero. Comparisons of the frequency distributions of labeled micronuclei with those of micronuclear "blanks" showed no evidence of a small population of labeled nuclei such as might be expected if micronuclei synthesized RNA for only a brief portion of the cell cycle. We conclude from these studies that there is no detectable RNA synthesis in Tetrahymena micronuclei during vegetative growth and reproduction.     

Macro- and Micronuclei of Tetrahymena pyriformis: A Model System for Studying the Structure and Function of Eukaryotic Nuclei*†

The macro- and micronucleus of Tetrahymena pyriformis are formed from a common diploid synkaryon during conjugation. Shortly after the 2nd postzygotic division, distinct morphologic and physiologic differences develop between the 2 nuclei. Micronuclei remain small, presumably diploid, and electronmicroscopic observations indicate that micronuclear DNA is contained in a dense, fibrous, chromosome-like coil. Macronuclei contain considerably more DNA than micronuclei, and the DNA of the macronucleus is found largely in the chromatin bodies typical of ciliate nuclei. The functional differences between macro- and micronuclei in vegetative cells also are striking. The template activity of DNA in the micronucleus is highly restricted compared to that in the macronucleus. Micronuclei synthesize and contain little RNA, and do not contain either nucleoli or ribonucleoprotein granules. Macronuclei, on the other hand, synthesize and contain large amounts of RNA and have many nucleoli and ribonucleoprotein granules. Macro- and micronuclei also have distinct differences in the timing of DNA synthesis during the cell cycle and in the timing and mechanism of nuclear division. Finally, during conjugation the macronucleus becomes pycnotic and disappears while the micronucleus undergoes meiosis and fertilization, ultimately giving rise to new macro- and new micronuclei. In short, the macro- and micronuclei of Tetrahymena provide an excellent system for studying the molecular mechanisms by which the same (or related) genetic information is maintained in different structural and functional states. Methods have been devised to isolate and purify macro- and micronuclei of Tetrahymena in the hope of correlating differences in the nucleoprotein composition of these nuclei with differences in their structure and function. The DNAs of macro- and micronuclei have been found to differ markedly in their content of a methylated base, N⁶-methyl adenine, and major differences in the histones of the 2 nuclei have been observed. Macronuclei contain histones similar to those found in vertebrate nuclei, while 2 major histone fractions seem to be missing in micronuclei. In addition, histone fraction F2A1 which is found in multiple, acetylated forms in macronuclei, is present only as a single, unacetylated form in micronuclei.     

Comparison of the sequences of macro- and micronuclear DNA of Tetrahymena pyriformis

Macro- and micronuclei were isolated from Tetrahymena pyriformis (Syngen 1, strain WH-6) and their DNAs compared by isopycnic centrifugation in neutral and alkaline CsCl, by analysis of thermal denaturation properties and by molecular hybridization. Unlike the situation observed in Stylonychia the buoyant densities and thermal denaturation patterns of Tetrahymena macro- and micronuclear DNAs were virtually identical—the only observable differences bordering on the limits of resolution of these techniques. DNA was isolated from the two nuclei which had been labelled with different radioactive isotopes (i.e. ¹⁴C-thymidine and ³H-thymidine), and the renaturation kinetics of mixtures of macro- and micronuclear DNA were examined using a single-strand specific deoxyribonuclease (S₁). Renaturation kinetics obtained using varying ratios of macro- and micronuclear DNA suggested that 80–90% of the sequences present in micronuclei were present in similar amounts in macronuclei. However, careful analyses of the renaturation kinetics indicate that approximately 10–20% of the sequences found in micronuclei are probably absent in macronuclei, and that most of these sequences are probably moderately repetitive (100 copies per genome or less). These findings place severe constraint on possible models concerning the structure of the Tetrahymena macronucleus, and are very different from the situation observed in Stylonychia where it has been suggested that only a small percentage of the sequences in micronuclei are present in significant amounts in macronuclei. Nonetheless, these results along with those in Stylonychia can be taken as an indication that the loss or under-replication of some DNA sequences accompanies macronuclear differentiation in ciliates.     

Qualitative and Quantitative Studies on DNA and RNA Synthesis in Loxodes striatus Nuclei*

SYNOPSIS. In this study the characteristics of the synthesis of DNA and RNA in the nuclei of Loxodes were investigated. Loxodes striatus is a primitive ciliate with 2 pairs of structurally differentiated diploid nuclei, the macro- and micronuclei. The macronuclei are differentiated morphologically into a clearly recognizable central core and an outer zone. To determine DNA and RNA synthesis, individual organisms were analyzed by autoradiography after incubating groups of cells with a ³H-labeled precursor ([³H]thymidine for DNA and [³H]uridine for RNA). The following observations were made: (A) All portions of macro- and micronuclei appeared to contain DNA as judged by the localizations of incorporated [³H]thymidine. (B) The macro- and micronuclei did not synthesize DNA at the same time; moreover, the duration of DNA synthesis in the former was much longer than of the latter nucleus. (C) Replication of DNA in the inner core and outer zone of the macronucleus occurred at separate times with little if any overlap. (D) All of the detectable [³H]uridine incorporation was found in the macronucleus and none in the micronucleus. Within the macro-nucleus the central core was more heavily labeled. (E) The quantitative differences in the label of the different components of the nuclear complex were investigated. (F) Contrary to the previously reported information our results suggest that DNA synthesis can occur in adult macronuclei. The possible explanation of these results is discussed in the context of the nuclear evolution of ciliates and of recent information on nuclear differentiation.     

Isolation and biochemical characterization of nuclei from chick embryo liver

A procedure is described for the isolation of enzymatically active nuclei from chick embryo liver. It consists of the homogenization of the pooled tissue in 0.32 M sucrose-3 mM MgCl₂ followed by a slow centrifugation. The resulting nuclear pellet is then purified further in a discontinuous density gradient composed of sucrose solutions containing Mg²⁺ ions, the lower portion of the gradient being 2.2 M sucrose-1 mM MgCl₂. Based on DNA recovery, the nuclear fraction isolated by the procedure described contained an average of 62% of the nuclei in the original filtered homogenate. Light and electron microscope examinations showed that 90% of the isolated nuclei were derived from hepatocytes. They appeared intact with well preserved nucleoplasmic and nucleolar components, nuclear envelope, and pores. The isolated nuclei were quite pure, having a very low level of cytoplasmic contamination as indicated by cytoplasmic enzyme marker activities and electron microscope studies. The nuclear fraction consisted of 19.9% DNA, 6.2% RNA, 74% protein, the average RNA/DNA ratio being 0.32. Biosynthetic activities of the two nuclear enzymes NAD-pyrophosphorylase and DNA-dependent RNA polymerase were preserved. The specific activities of these enzymes were: NAD-pyrophosphorylase, 0.049 µmoles nicotinamide adenine dinucleotide (NAD) synthesized/min per mg protein; Mg²⁺ activated RNA polymerase, 4.3 µµmoles UMP-2-C¹⁴ incorporated into RNA/µg DNA per 10 min; and Mn²⁺-(NH₄)₂SO₄ activated RNA-polymerase, 136 µµmoles UMP-2-C¹⁴ incorporated into RNA/µg DNA per 45 min.     

DNA Damage and Repair in Stylonychia lemnae (Ciliata,Protozoa)1

ABSTRACT Irradiation with X rays, UV irradiation after incorporation of bromodeoxyuridine (BU) into the DNA, and cis-platinum (cis-Pt) treatment each cause the loss of micronuclei of Stylonychia lemnae while the macronuclei are not severely affected. The abilities of both nuclei to repair DNA were investigated. Unscheduled DNA synthesis could not be demonstrated after X-ray irradiation, but it was found after treatment with BU/UV and cis-Pt in macro- and micronuclei. The extent of the repair process in the micro- and macronuclei was alike, as indicated by grain counts of [6-³H]thymidine-treated cells. One reason for the different sensitivity of both nuclei to DNA-damaging treatment may be the different number of gene copies in the macro- and micronuclei.     

Numbers of 5S and tRNA genes in macro- and micronuclei of Tetrahymena pyriformis

Macronuclei of Tetrahymena pyriformis contain approximately 200 copies of the genes for 25S and 17S ribosomal RNA (rRNA) per haploid genome. Micronuclei, however, contain only a few copies of the rRNA genes per haploid complement. Since macronuclei develop from, products of meiosis, fertilization and division of micronuclei, we suggested that the multiple copies of the rRNA genes in macronuclei are generated by amplification of the small number of genes in micronuclei (Yao et al., 1974). This process provides a simple mechanism for maintaining the homogeneity of the repeated rRNA genes. To test if amplification is a general mechanism operating on all repeated genes in Tetrahymena, we have examined the numbers of 5S RNA and tRNA genes in macro- and micronuclei. 5S RNA was purified by polyacrylamide gel electrophoresis and hybridized to saturation against macro- and micronuclear DNA. Approximately 0.013–0.014% of macronuclear DNA and about 0.009% of micronuclear DNA is complementary to 5S RNA. After correcting for the differences in the DNA sequence complexities between the two nuclei, we calculate that there are 300–350 5S genes per haploid macro- or micronuclear genome. From these data we conclude that there is little or no detectable amplification of the 5S genes in macronuclei relative to micronuclei. Similar studies using tRNA indicate that these genes are also highly repeated in both nuclei; about 800 genes are present per haploid genome. Thus, amplification from a small number of genes can be excluded as the mechanism for generating the repeated copies of the 5S and tRNA genes in Tetrahymena and it is likely that another, as yet unidentified, mechanism operates to maintain the homogeneity of these genes.     

Isolation and Properties of Nuclei from Control and Auxin-treated Soybean Hypocotyl

A quick procedure for the isolation of nuclei with good yield from soybean hypocotyl (Glycine max var. Wayne) was developed. The isolated nuclei appeared to retain their structural integrity. They were typically ellipsoidal with minima and maxima diameter of about 6 and 8 to 10 micrometers. While the nuclei were similar in size, the nucleoli were significantly larger in nuclei from auxin-treated tissue. The DNA content per nucleus was 4 ± 1 picograms for both untreated and auxin-treated tissues. The DNA: RNA: protein ratio of isolated nuclei in untreated and auxin-treated tissues was 1: 3.1: 11 and 1: 5.4: 21.7, respectively. The purified nuclei were active in RNA synthesis; the level of RNA polymerase II activity expressed in the nuclei from untreated tissue was 50 to 60% higher than RNA polymerase. I. The nuclei from auxin-treated tissues contained about 2.5 times as much RNA polymerase I activity as nuclei from untreated tissue. The purified nuclei from both untreated and auxin-treated tissues were also active in the incorporation of ³H-TTP into DNA.     

SYNTHETIC CAPACITIES OF CHROMOSOME FRAGMENTS CORRELATED WITH THEIR ABILITY TO MAINTAIN NUCLEOLAR MATERIAL

Onion (Allium cepa) and bean (Vicia faba) root tip cells containing many micronuclei, derived from x-ray-induced chromosome fragments, were exposed to H³-thymidine and H³-cytidine to determine the ability of such fragments to undergo DNA and RNA synthesis. Only a few micronuclei in onion and many in bean roots synthesize nucleic acid simultaneously with their main nuclei. A few micronuclei labeled with H³-thymidine undergo mitotic chromosome condensation along with the main nuclei, while the unlabeled ones never do so. The onset of nucleic acid synthesis as well as mitosis in micronuclei appears to be under generalized cellular control. Although all chromosomes and chromosome fragments at telophase give a positive reaction for a silver stainable nucleolar fraction, in the subsequent interphase only some micronuclei, derived from such chromosome fragments, are found to maintain nucleoli; others lose them with time. Those micronuclei which maintain nucleoli, perhaps due to the presence of specific chromosomal regions, are also active in DNA and RNA synthesis. These results are compatible with the concept that nucleoli and associated chromosome regions play an important role in the primary biosynthetic processes of the cell.     

Characterization of Bacteriophage S13 suN15 Single-Strand and Replicative-Form Deoxyribonucleic Acid

The deoxyribonucleic acid (DNA) of bacteriophage S13 was shown to be single-stranded by the criteria of reactivity with formaldehyde, dependence of optical density on ionic strength, broad temperature-absorbance profile, and lack of molar equivalence of the purine and pyrimidine bases. The DNA has a molecular weight of 1.8 × 10⁶ daltons, an S°₂₀ of 24.6 in SSC (0.15 m NaCl plus 0.015 m sodium citrate), and a buoyant density of 1.726 g/cc in CsCl. Electron microscopy showed the molecule to be circular. S13 replicative-form DNA was shown to be a double-stranded, circular molecule with a molecular weight of 3.5 × 10⁶ daltons, an S_[ill] of 20.7 in SSC, and a buoyant density in CsCl of 1.710 g/cc. The finding that S13 DNA is slightly more pyrimidine-rich than X174 DNA but is indistinguishable by all other parameters supports the close genetic relationship between the two bacteriophages.     

Organization of the 5S RNA genes in macro- and micronuclei of Tetrahymena pyriformis

The organization of the 5S genes in macro- and micronuclei of Tetrahymena pyriformis was studied using restriction endonucleases. After complete digestion of macronuclear DNA with BamH-I or Hpa I, 5S RNA hybridized to a DNA fragment of approximately 280 base pairs (bp). When macronuclear DNA was only partially digested with these enzymes, hybridization with ³²P-5S RNA demonstrated an oligomeric series with a spacing of 280 bp. These results indicate that the 5S genes are tandemly repeated in macronuclei and that the repeating unit is 280 bp (or 180,000 daltons). Since 5S RNA is 120 nucleotides, we conclude that the 5S repeat units contain a 120 bp transcribed region and a 160 bp spacer region. When macronuclear DNA was digested with Eco RI, Bgl I, or Eco RI + Bgl I, 5S RNA hybridized to DNA of molecular weight 3–4×10⁶, suggesting that these enzymes do not cleave within a 5S repeat. These 3–4×10⁶ dalton fragments define the maximum size of an average cluster of 5S repeated units. Assuming the size of the 5S repeat to be 0.18×10⁶ daltons, there are about 15–20 5S repeats per average tandem cluster, and since there are 350 5S-genes per haploid genome, there must be approximately 15–20 tandem arrays. Results obtained using micronuclear DNA suggest that organization of the 5S-genes is very similar in macro- and micronuclei. Macronuclear rRNA genes are extracnromosomal palindromic dimers. In contrast, 5S genes in Tetrahymena were found to be integrated within the genomes of both macro- and micronuclei and not linked to the rRNA genes. Moreover, it is unlikely that they are palindromes; rather they appear to be tandemly repeated in head-to-tail linkages. Thus, the organization of the 5S genes in Tetrahymena is similar to that of higher eukaryotes.     

Isolation and preliminary characterization of cryptic satellite DNA in pea

Optimum conditions have been established for isolation of ‘cryptic’ satellite DNA from the genome of pea (Pisum sativum), using gradients of CS₂SO₄ containing silver ions. At an Ag⁺ :DNA-P ratio (R) of 0.1, and at alkaline pH, four fractions are obtained: mainband (buoyant density 1.437 g cm³; 67% of total DNA), satellite I (buoyant density 1.582 g/cm³; 7% of total DNA), satellite II (buoyant density 1.520 g/cm³, 11% of total) and satellite III (buoyant density variable between 1.45 and 1.51 g/cm³; 15% of total). The reiterated DNA content of these four fractions has been investigated by reassociation experiments conducted over a Cot range of 1 × 10^?5 to 2.0. All four fractions contain at least two kinetic components; each fraction, including the mainband, consists at least partly of highly reiterated DNA. Ribosomal RNA hybridizes only to the mainband.     

Qualitative and quantitative studies on DNA and RNA synthesis in Loxodes striatus nuclei.

In this study the characteristics of the synthesis of DNA and RNA in the nuclei of Loxodes were investigated. Loxodes striatus is a primitive ciliate with 2 pairs of structurally differentiated diploid nuclei, the macro- and micronuclei. The macronuclei and differentiated morphologically into a clearly recoginzable central core and an outer zone. To determine DNA and RNA synthesis, individual organisms were analyzed by autoradiography after incubating groups of cells with a 3H-labeled precursor ([3H]thymidine for DNA and [3H]uridine for RNA). The following observations were made: (A) All portions of macro- and micronuclei appeared to contain DNA as judged by the localizations of incorporated [3H]thymidine. (B) The macro- and micronuclei did not synthesize DNA at the same time; moreover, the duration of DNA synthesis in the former was much longer than of the latter nucleus. (C) Replication of DNA in the inner core and outer zone of the macronucleus occurred at separate times with little if any overlap. (D) All of the detectable [3H]uridine incorporation was found in the macronucleus and none in the micronucleus. Within the macronucleus the central core was more heavily labeled. (E) The quantitative differences in the label of the different components synthesis can occur in adult macronuclei. The possible explantion of these results is discussed in the context of the nuclear evolution of ciliates and of recent information on nuclear differentiation.     

Binding of the chemical carcinogen N-hydroxy-acetyl-aminofluorene to ploidy classes of rat liver nuclei as separated by velocity sedimentation at unit gravity

A large sedimentation device was developed that allows separation of 5 × 10⁸ rat liver nuclei by velocity sedimentation at unit gravity. Using the apparatus isolated rat liver nuclei were separated into classes of diploid stromal (Von Kuppfer, sinusoidal lining) nuclei, diploid parenchymal nuclei and tetraploid parenchymal nuclei respectively. DNA content and volume of the nuclei were measured. Diploid nuclei were 100% pure; tetraploid nuclei 98%.The in vivo binding of the liver carcinogen [³H]-N-hydroxy-AAF to these classes of nuclei was determined (total binding to protein, DNA and RNA). Binding and the subsequent removal of the fluorene derivatives was registered as a function of time. At all stages diploid stromal nuclei bound 2.6–5 times less carcinogen than did diploid parenchymal nuclei. Tetraploid parenchymal nuclei bound more than twice (2.3–3.95) the amount, that was present in their diploid counterpart. This effect became more pronounced 11 days after application of N-hydroxy-N-acetyl-2-aminofluorene.DNA was enzymatically purified from pooled classes of the various nuclear types. For purified DNA also it was found that DNA derived from diploid stromal nuclei bound 2.6–2.8 times less carcinogen than did DNA derived from diploid parenchymal nuclei.     

DNA SYNTHESIS IN NEURONAL, GLIAL AND LIVER NUCLEI ISOLATED FROM THE ADULT GUINEA PIG

Abstract— [³H]Deoxythymidine-5′-triphosphate incorporation into P₅₁ (51% neuronal nuclei: 49% glial nuclei), P₃ (3% neuronal nuclei: 97% glial nuclei) and liver nuclear preparations, isolated from the adult guinea pig, was determined in the presence of the other three complementary deoxyribonucleo-tides. The enzymic characteristics of the DNA synthesis reaction were studied and DNA polymerase contents were estimated in neuronal, glial and liver nuclei. (1) Cerebral and liver nuclei exhibited similar enzymic properties for DNA synthesis activities with a few discrepancies. (2) P₅₁ nuclei synthesized DNA 2.4-fold more actively than P₃ nuclei. Liver nuclei carried out the most active DNA synthesis. The proportion of chromatin DNA available as template and primer was estimated by comparison with native calf thymus DNA. The available proportions found, in terms of the total chromatin DNA. were 2.39% for P₅₁ nuclei, 1.38% for P₃ nuclei and 37.6% for liver nuclei. (3) Exogenous native and heat-denatured calf thymus DNA were utilized as template and primer by DNA polymerase in nuclei in different ways depending on the nuclear species. The enzyme was saturated with native DNA by elevating the concentration and the activity reached a plateau. Denatured DNA inhibited the activity at the higher concentrations. (4) From the enzyme activities at a saturation concentration of exogenous DNA, DNA polymerase contents were estimated: P₅₁ nuclei, 39.2 ± 2.6 (s.e.m. ) units (fmol of TMP incorporated/30 min at 31°C)/μg of nuclear DNA; P₃ nuclei. 24.5 ± 1.6; and liver nuclei, 72.5 ± 8.1; the specific activity obtained on a protein basis was 1.55 times higher with P₃ nuclei than with P₅₁ nuclei. (5) Denatured DNA inhibited the nuclear DNA polymerase activity dependent on native DNA. The efficiency of inhibition was in the order: P₃ > P₅₁ > liver nuclei.     

Efficient Plant Genomic and Viral DNA Isolation from Mature Leaf Midrib of Banana (Musa spp)

The genomic DNA isolation from mature leaf midrib is a tough job, because of the abundance of polysaccharides and secondary metabolites, which interferes with DNA isolation as well as polymerase chain reaction (PCR) studies. The leaf midrib of 3^rd leaf from 3-moths old, ex-vitro developing banana [AAA, Dwarf Cavendish-Basrai (Sindhri banana)] plants (healthy and BBTV infected) was grinded in liquid N₂. Exact 0.3 g of leaf midrib powder was washed with washing buffer (100 mM Tris-Cl, 5 mM EDTA, 0.35 M sorbitol, 1% 2-mercaptoethanol) then homogenized in 0.8 ml of three different pre-heated (60°C) DNA isolation buffers. Supernatant was extracted through phenol: chloroform:isoamyl alcohol (25:24, v/v), chloroform: isoamyl alcohol (24:1, v/v) and finally with chloroform (100%) one by one. Maximum yields were ranged from 49.33 and 27.73 μg mg ^?1 DNA with impurities 5.67 and 5.87 μg mg^?1 through buffer I, while 45.77 and 25.53 μg mg^?1 DNA with 6.13 and 6.16 μg mg^?1 impurities through buffer III from healthy and infected plants respectively. Best one RAPD was observed in all the DNA samples isolated with different buffers, while viral amplification was good in DNA isolated with buffer I and II, when 10 (RAPD) and 25 ng DNA (C ₁ gene) was used as a template in a reaction of 25 μl. Meanwhile, buffer II is limited for viral DNA isolation while buffer I (1M Tris-Cl, 5M NaCl, 2 % cTAB, 50mM EDTA, 1 % PVP, 0.2 % 2-mercaptoethanol) has dual capacity for plant and virus DNA isolation. This described protocol is economic in terms of times, labor and cost.     

The ribosomal cistrons of the water mold Achlya bisexualis

Total DNA was extracted from vegatative mycelia of the water mold Achlya bisexualis. Fractionation of the DNA in CsCl gradients resulted in two components: a major component with a buoyant density of 1.697 g cm^?3 and a minor component with a density of 1.685 g cm^?3. The minor component has been identified as mitochondrial DNA based on extractions from isolated mitochondria and Triton X-100 washed nuclei. Detergent washing of the nuclei yielded DNA in which the mitochondrial DNA component was absent, while the isolated mitochondrial preparations contained DNA enriched in the 1.685 g cm^?3 component. Hybridization studies of A. bisexualis DNA to rRNA show that the ribosomal cistrons have a buoyant density coincident with that obtained with the nuclear DNA. In addition, preliminary evidence indicates that the mitochondrial DNA does not hybridize to the cytoplasmic RNA under the conditions used for this study. Ribosomal RNA hybridized to about 0.65% of the total DNA.     

DIRECT COUNTING AND SIZING OF MITOCHONDRIA IN SOLUTION

Resistive particle counting has been developed for the accurate sizing and counting of mitochondria in solution. The normal detection limit with a 30 µ aperture is 0.48 µ diameter, or 0.056 µ³ particle volume The mean volume of rat liver mitochondria was 0.42 µ³ or 0.93 µ in diameter. The average value for numbers of particles per milligram of mitochondrial protein was 4.3 x 10³, and per gram of rat liver was about 11 x 10¹⁰. These values compare satisfactorily with those derived by light microscopy and electron microscopy. The mean volume for mitochondria from rat heart was 0 60 µ³ and from rat kidney cortex, 0.23 µ³. These values agree within 15% of those determined by electron microscopy of whole tissue. Mitochondrial fragility and contaminating subcellular organelles were shown to have little influence on the experimentally determined size distributions The technique may be applied to rapid swelling studies, as well as to estimations of the number and size of mitochondria from animals under different conditions such as liver regeneration and hormonal, pathological, or drug-induced states Mitochondrial DNA, RNA, cytochrome c-oxidase, cytochrome (a ÷ a₃), and iron were nearly constant per particle over large differences in particle size. Such data may be particularly valuable for biogenesis studies and support the hypothesis that the net amount per particle of certain mitochondrial constituents remains constant during mitochondrial growth and enlargement