Specific fragmentation of mitochondrial DNA from Neurospora crassa by restriction endonuclease Eco R I.

Linear, size-heterogenous mitochondrial DNA from $\text{Neurospora crassa}$ was cleaved by the restriction endonuclease Eco R I into eleven specific fragments. According to their contour lengths the fragments have molecular weights between 1.1 and 14 × 10⁶. The sum of the fragments lengths is identical with the contour length (19.8 μm, 41 × 10⁶ daltons) of the few circular molecules detectable in purified DNA preparations.The results suggest sequence homogeneity of mitochondrial DNA and further demonstrate that restriction enzymes can be used to establish a physical map of an unspecifically-fragmented DNA molecule.     

Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures.

Double-stranded DNA fragments terminated at their 5′-ends by the singlestranded sequence pA-A-T-T-, generated by digestion of DNA with EcoRI restriction endonuclease, were ligated with Escherichia coli polynucleotide ligase under various conditions of temperature, concentration and time. The linear and circular products of ligation were separated by electrophoresis in agarose gel and quantitated by densitometry. The rate of ligation of (EcoRI-cleaved) simian virus (SV40) DNA at a concentration of 100 μg/ml increased from 0 °C to 5 °C to 10 °C (6-fold increase overall); raising the temperature to 15 °C did not further increase the rate of ligation. At the appropriate DNA concentrations, the predominant products of ligation are either linear concatemers that are integral multimers of the starting DNA fragment, or covalently closed circular structures of the monomeric DNA fragment. Ligating a mixture of two different length DNA fragments gives rise to all of the possible expected recombinant molecules.Linear or circular products of ligation were predicted by consideration of the total concentration of DNA termini, i, and the local concentration of one terminus in the neighborhood of the other on the same DNA molecule, j. The parameter j is a function of the length of a DNA molecule, providing this length is greater than the random coil segment of DNA. Experimentally it was found that circular structures are formed in significant amounts only under conditions when the value of j is several times greater than that of i. When j = i, equal amounts of linear and circular products would be expected, but most of the molecules were ligated into linear concatemers. No circular structure of a DNA fragment whose contour length l (6 × 10⁻² μm) is smaller than the random coil segment value b (7·17 × 10⁻² μm) was observed, while circular structures of the dimer of the same molecule (12 × 10⁻² μm) were detected.     

Synthesis of satellite DNA in Rhynchosciara hollaenderi

During the latter part of the fourth instar development of Rhynchosciara larvae, DNA synthesis occurs in both germ cells and somatic cells, even though these cells do not undergo mitosis. CsCl density gradient analysis has revealed the synthesis of d(AT) satellite DNAs in the testis and in somatic tissues such as salivary gland, fat body, and gastric cecum. In these studies it has been shown that there is a tissue-specific variation in the relative proportions of synthesis of d(AT) satellite and main-band DNA in the testis during the fourth instar. The initiation of synthesis of the d(AT) satellite in the testis corresponds with the appearance and formation of the highly characteristic mitochondria which develop during the maturation of the spermatocytes. This satellite DNA has been shown to be circular and has a density of 1.681 gm/cm³ in CsCl. Synthesis of a similar circular DNA cannot be detected in somatic tissues, although these tissues do synthesize a d(AT) satellite of similar density.     

STUDIES ON MITOCHONDRIA : II. Mitochondrial DNA of Thoracic Muscles of Schistocerca gregaria

The buoyant densities of the nuclear and mitochondrial DNA from the thoracic muscles of Schistocerca gregaria were found to be 1 702 and 1.689 g/cm³, respectively, corresponding to guanine plus cytosine (G + C) content of 42.2 and 30% A preliminary treatment of the mitochondrial pellet with DNase (25°C, 20 min) is necessary to eliminate the contaminating nuclear DNA. The mitochondrial DNA renatures readily after heat denaturation and incubation at 65°C. The DNA released from the mitochondrial pellet by osmotic shock consists of circular open and closed molecules with a contour length around 5 µ The instability of insect mitochondria in in vitro preparations is discussed.     

Shape of the ciliary doublet microtubule in solution

The individual doublet microtubule (DMT) from Tetrahymena cilia had the appearance of a circular arc in solution and its contour length was around 5 to 6 μm as observed under a dark-field microscope. One end of the circular arc was exactly in focus on the slide under the microscope, whereas the other end was slightly out of focus, suggesting that the circular arc may be regarded as a part of a coiled helix. On measuring the contour length and the end-to-end distance of the arc in solution, we found that the Ca-induced transformation of the ciliary DMT took place at a Ca concentration of about 10^?6m in the medium; that is, the radius of the arc was 3.4 ± 0.9 μm at 10^?3m-Ca and 1.5 ± 0.1 μm at less than 10^?6m-Ca. Change in the pH of the medium brought about no significant difference in the radius of the arc of the DMT. The individual DMT of a negatively stained sample appeared to have a configuration similar to that of the circular arc in solution, bending at right angles to the partition wall between the A and B-tubules. Based on the criterion that the A-tubule is wider than the B-tubule, we found that the A-tubule occurred on the outer side of the circular arc in 90 to 100% of the samples.     

Recombinant DNA analysis indicates that the multiple chromosomes of maize mitochondria contain different sequences

Twenty-eight Bam H 1 restriction fragments were isolated from normal mitochondrial DNA of maize by recombinant DNA techniques to investigate the organization of the mitochondrial genome. Each cloned fragment was tested by molecular hybridization against a Bam digest of total mitochondrial DNA. Using Southern transfers, we identified the normal fragment of origin for d each clone. Twenty-three of the tested clones hybridized only to the fragment from which the clone was derived. In five cases, labeling of an additional band indicated some sequence repetition in the mitochondrial genome. Four clones from normal mitochondrial DNA were found which share sequences with the plasmid-like DNAs, S-1 and S-2, found in S male sterile cytoplasm. The total sequence complexity of the clones tested is 121×10⁶ d (daltons), which approximates two thirds of the total mitochondrial genome (estimated at 183×10⁶ d). Most fragments do not share homology with other fragments, and the total length of unique fragments exceeds that of the largest circular molecules observed. Therefore, the different size classes of circular molecules most likely represent genetically discrete chromosomes in a complex organelle genome. The variable abundance of different mitochondrial chromosomes is of special interest because it represents an unusual mechanism for the control of gene expression by regulation of gene copy number. This mechanism may play an important role in metabolism or biogenesis of mitochondria in the development of higher plants.     

Oligosaccharides of human milk. Isolation and characterization of three new disialyfucosyl hexasaccharides.

Renal mitochondrial 25-hydroxyvitamin D₃-1-hydroxylase (1-hydroxylase) is sensitive to inhibition by 2 × 10^?5m calcium and 5 × 10^?3m phosphate when hydroxylation is supported by either malate or NADPH. This sensitivity to ion inhibition is observed in mitochondria from both vitamin D-deficient and repleted chicks and remains when mitochondria are frozen and thawed or are incubated in a hypotonic medium. The ionophore A23187 inhibits the 1-hydroxylase but partially reverses the inhibition exerted by 2, 5, or 7.5 × 10^?5m calcium. Addition of a kidney soluble cell fraction (37,000g supernatant) to isolated mitochondria did not enhance the 1-hydroxylase activity under conditions of varied substrate concentration, osmolarity of the incubation medium, or mitochondrial washes. It is concluded that a soluble cellular component is not involved in the regulation of the 1-hydroxylase but that intramitochondrial calcium and phosphate may well play a role in its regulation.     

Isolation and characterization of DNA from the entomopathogenBeauveria bassiana

Beauveria bassiana chromosomal (chr) and mitochondrial (mt) DNA were isolated, purified, and characterized. Modification of a recent rapid method of total DNA extraction was followed by DNA purification on CsCl-bisbenzimide gradients. Cytosine methylation at 5′-CpG-3′ doublets was undetectable in the total DNA as demonstrated byMspI andHpaII restriction enzyme digests. The chrDNA had a bouyant density of 1.7130 g/ml (G + C ratio of 53.4%) and a T_m of 92.6°C (G + C ratio of 56.9%). Mitochondrial DNA was isolated from total DNA or purified mitochondria and had a buoyant density of 1.7120 g/ml (G + C ratio of 52.3%) and a biphasic DNA melting curve. A minor melt at 76.2°C (16.8% G + C ratio) yielded a 10% increase in absorbance while the major melt at 93.1°C (58% G + C ratio) yielded a 30% increase in absorbance. The mtDNA was estimated by gel electrophoresis to be 17.6 ± 0.6 MDa and determined to be 28.5 kb by restriction enzyme analysis. Electron microscopy of the mtDNA revealed circular molecules having an average contour length of 8.84 ± 0.51 μm. A physical map of the mtDNA was generated by double restriction enzyme digestion using the restriction enzymesClaI,EcoRI,SalI,BstEII, andXhoI.     

Measurement of mitochondrial oxidative phosphorylation: Selective inhibition of adenylate kinase activity by P1,P5-di-(adenosine-5′)-pentaphosphate

A biochemical assay for the measurement of ATP synthesis coupled to electron transport in the presence of adenylate kinase was developed as an alternative to using the conventional Clark-type oxygen electrode. The assay utilizes P¹,P⁵-di-(adenosine-5′)-pentaphosphate which is shown to be a competitive inhibitor with MgADP for rat liver mitochondrial adenylate kinase (K_i = 7.04 × 10^?8m) and was found to have no effect on oxidative phosphorylation of either intact mitochondria or submitochondrial particles.     

Structure and physico-chemical properties of bacteriophage G: III. A homogeneous DNA of molecular weight 5 × 108

The physico-chemical properties of the DNA released from bacteriophage G (active on Bacillus megatherium) are described. Phage G, an unusually large bacteriophage, has a nucleic acid content of 4 to 6 × 10⁸ daltons.Sedimentation velocity analysis at low angular speed and examination by electron microscopy, indicate that a single DNA molecule, sedimenting with s_{20, w}⁰ = 125 ± 1.5 S and at least 200 ± 20 μm long, is released upon thermal or osmotic shock. Melting temperature data and chromatographic analysis indicate a mean base composition of 70% A + T. CsCl and Cs₂SO₄ buoyant density data, circular dichroism spectra and sensitivity to specific nucleases indicate that phage G DNA is similar to the DNAs from T-even phages and is more glucosylated than phage T6 DNA. Direct glucose determination indicates a 185% molar ratio of glucose to cytosine. Linear density extrapolated from literature data and contour length measurement yield a lower limit for the molecular weight of phage G DNA of 4.9 × 10⁸. Comparison of this value with the s_20,w⁰ measured with the analytical ultracentrifuge seems to confirm the validity of the empirical relationship proposed by Freifelder (1970), between s_{20, w}⁰ and molecular weight, over a larger range than that previously known. A possible systematic error in defect in length determination, however, prevents a discrimination between this and other empirical formulae proposed by various authors, which predict a molecular weight that is 20 to 25% higher.     

On the molecular length of the replicative form DNA of bacteriophage phiX174

This paper describes an electron microscopic study of the circular replicative form DNA of bacteriophage φX174. The study has been carried out using a preparative technique in which the DNA molecules are adsorbed from solution on to the cleavage surface of mica and visualized in the electron microscope as a metal-shadowed replica (Gordon &; Kleinschmidt, 1969,1970). Contour lengths of open circular molecules were measured in samples obtained from preparations in which the following experimental parameters were varied: the ionic strength of the solution from which the DNA was adsorbed on the mica and the way in which the molecules were dried before shadowing. At the 0.05 significance level, varying these parameters had no effect on the mean length and variances of samples of molecules obtained from five experiments; the samples were therefore regarded as being drawn from the same molecular population with a mean length and variance of, respectively, 1.83 μm and 0.0117 μm².It was argued that the DNA molecules adsorbed on the mica are “frozen” into the molecular conformation present in solution at the time of adsorption and that, therefore, the experimentally determined contour lengths represent authentic molecular lengths in solution. Based on current estimates of the replicative form DNA molecular weight, the mean contour length obtained was slightly but significantly larger than the length predicted for molecules in an exact B configuration. The variance was larger than could be attributed solely to experimental error, indicating that the molecular population in aqueous solution is heterogeneous in contour length. These experimental results were shown to be consistent with a model for DNA structure in aqueous solution in which individual molecules are dynamic variants of a perturbed B form structure (von Hippel &; Wong, 1971).     

Ribosomal RNA synthesis in mitochondria of Neurospora crassa

Ribosomal RNA synthesis in Neurospora crassa mitochondria has been investigated by continuous labeling with [5-³H]uracil and pulse-chase experiments. A short-lived 32 S mitochondrial RNA was detected, along with two other short-lived components; one slightly larger than large subunit ribosomal RNA, and the other slightly larger than small subunit ribosomal RNA. The experiments give support to the possibility that 32 S RNA is the precursor of large and small subunit ribosomal RNA's. Both mature ribosomal RNA's compete with 32 S RNA in hybridization to mitochondrial DNA. Quantitative results from such hybridization-competition experiments along with measurements of electrophoretic mobility have been used to construct a molecular size model for synthesis of mitochondrial ribosomal RNA's. The large molecular weight precursor (32 S) of both ribosomal RNA's appears to be 2.4 × 10⁶ daltons in size. Maturation to large subunit RNA (1.28 × 10⁶ daltons) is assumed to involve an intermediate ~1.6 × 10⁶ daltons in size, while cleavage to form small subunit RNA (0.72 × 10⁶ daltons) presumably involves a 0.9 × 10⁶ dalton intermediate. In the maturation process ~22% of the precursor molecule is lost. As is the case for ribosomal RNA's, the mitochondrial precursor RNA has a strikingly low G + C content.     

Late spermatocytes from immature rat testis isolation,electron microscopy,lectin agglutinability and capacity for glycoprotein and sulfogalactoglycerolipid biosynthesis

A method is described for preparing late spermatocytes form immature rat testes which yields about 2 · 10⁵ cells per testis a purity of 70–80% and a viability of over 90%. The spermatocytes are highly agglutinable by both concanavalin A and wheat germ agglutinin but no major difference in lectin-mediated agglutinability was observed between late spermatocytes, early spermatids and spermatozoa. Isolated spermatocytes were capable of incorporating [¹⁴C]glucosmine into glycoprotein at a linear rate for about 50 min at 30°C and contained a glycoprotein N-acetylglucosaminyltransferase (8.6 nmol/mg protein per h) and a glycoprotein fucosyltransferase (4.5 nmol/mg protein per h) previously described in partially purified adult mouse testicular germinal cells. A Golgi-rich fraction was prepared from isolated spermatocytes wchich was enriched 15-fold in the N-acetylglucosaminyltransferase, 19-fold in the fucosyltransferase and 3-fold in a galactosyltransferase. These studies showed that late spermatocytes were higly active in glycoprotein synthesis. Studies on the incorporation of [³⁵S]sulfate into sulfogalactoglycerolipid indicated that late spermatocytes were not the primary site of synthesis of this lipid although late spermatocytes were shown to be highly enriched in sulfogalactoglycerolipid (5 times the level in whole rat testis). Further, [³⁵S]sulfogalactoglycerolipid took 5 weeks to migrate from its site of synthesis to the epididymis. These studies suggest that sulfogalactoglycerolipid is sulfated at a spermatocyte cell stage prior to the late (pachytene and diplotene) seprmatocyte stage.     

The nuclear DNA content and chromatin ultrastructure of the coelacanth Latimeria chalumnae

By measurement of 731 erythrocytes by Feulgen cytophotometry, the nuclear DNA content of the coelacanth Latimeria chalumnae Smith is determined to be 7.22 picograms (pg). This value is high among fishes but is closely comparable to that of man and most other mammals. The average mass of erythrocyte nuclear chromatin, measured by quantitative electron microscopy, is 15.2 pg. This chromatin is in the form of fibers having a mean diameter of 202 Å. The average weight of the chromatin fiber is 6.75 × 10^?16 g/μm. Thus, the nucleus contains 22 500 μm of chromatin fiber. Dividing the nuclear DNA content of Latimeria by the known mass of the DNA double helix (3.26 × 10^?18 g/μm) gives a total length of 2 215 000 μm of DNA double helix. In comparing these two measurements of structural length, it is found that 98.4 lengths of double helix are packed into one length of chromatin fiber. This packing ratio is over three times greater than that of human G1 lymphocytes. The difference may be attributable to the difference between the two tissues and thus reflect a functional distinction, or it may be due to the difference between the two species and reflect an evolutionary distinction.     

Large circular mitochondrial DNA in Crithidia luciliae

A novel circular DNA, 11.3 μm in contour length, has been found in a pure kinetoplast DNA fraction of Crithidia luciliae. The mitochondrial nature of the kinetoplast and the absence of these large circular molecules in the nuclear fraction of DNA suggest that they constitute the mitochondrial genome of this species.     

Size and structure of mitochondrial DNA from Physarum polycephalum

One band of DNA with a buoyant density of 1.688 g cm⁻³ is found when isolated mitochondria of the slime mold Physarum polycephalum are incubated with deoxyribonuclease prior to lysis. The DNA consisted mainly of linear molecules up to about 20 μm in length. As many as 10% of the molecules were, however, of open circular conformation with a circumference of 19.1±0.5 μm. Mild lysis conditions favoured the isolation of DNA/protein complexes with no visible free ends. The data suggest that the mitochondrial DNA (mtDNA) of Physarum is a circle and that circularity may be maintained by DNA/protein interaction.     

Cloning of Escherichia coli genes encoding 3-methyladenine DNA glycosylases I and II

Summary The presence of polydisperse small circular DNAs in wheat cells was first confirmed by the mica-pressadsorption (MPA) method for electron microscopy. To identify their location in the cell, chloroplast and mitochondrial fractions were examined separately by the same method; small circular DNAs were scarcely found in the former but abundantly in the latter fraction, indicating their origin from mitochondria. The size varied greatly, ranging from 0.1 to 2.0 m in contour length. To verify the present finding, the same mitochondrial fraction was examined by the conventional cytochrome-spreading method by which the presence of the same size-class of circular DNAs was confirmed.To know the relationship between the small circular DNAs and cytoplasmic differentiation observed among Tritium (wheat) and Aegilops species, protoplasts isolated from seven alloplasmic lines of common wheat with different cytoplasms were examined by the MPA method. Similar polydisperse small circular DNAs, ranging from 0.1 to 2.5 m in contour length Dere found in all lines, and no clear size differences were noticed among the DNA populations from the cytoplasms of eight Triticum and Aegilops species.     

Transformation ofSchizophyllum commune: An analysis of specific properties

Several properties of transformation in the basidiomycete,Schizophyllum commune, were examined. The transformation efficiency of protoplasts made from germinating basidiospores is dependent upon the length of time that the spores are incubated under conditions that promote germination. Protoplasts prepared from ungerminated spores transform at least 10 times more efficiently than protoplasts prepared from germlings (25 μm in length) or from mycelium. Transformation frequencies of 1000 transformants/μg of control plasmid DNA and 10⁷ protoplasts are sufficient for obtaining transformants with 2 × 10⁷ protoplasts and 10 μg of bank DNA from a genomic plasmid library. The probability of cotransforming with two plasmids is dependent on the DNA concentrations of each; concentrations can be adjusted to yield nearly 100% cotrasformants. The presence of a nonselected plasmid in the reaction mix improves the transformation frequency of a selected marker carried on another plasmid; this is not true if linear fragments ofSchizophyllum genomic DNA are used as the nonselected DNA. Transformation of aSchizophyllum protoplast does not require its fusion to another protoplast.     

Replicating circular DNA molecules in yeast

The yeast Saccharomyces cerevisiae contains a class of small circular DNA molecules, approximately 2 μm in contour length (Sinclair et al., 1967). In this report, it is shown that these molecules replicate as double-branched circles, similar to those observed during replication of the bacteriophage λ and Escherichia coli chromosomes. A normal rate of replication of these DNA circles requires the function of a nuclear gene, cdc 8.