Regulation of ribonucleic acid synthesis in Escherichia coli B-r: an analysis of a shift-up. 1. Ribosomal RNA chain growth rates

The chain growth rate for ribosomal RNA was determined for Escherichia coli$\text{B}\text{r}$ growing in succinate (μ = 0.69 doublings/h), glucose (μ = 1.36) and glucose/ amino acids (μ = 2.10) medium. With increasing bacterial growth rate the chain growth rate increases from 4400 to 6300 nucleotides/min. These values are almost twofold higher than the chain growth rate reported for messenger RNA; this implies that, following a nutritional shift-up, the transfer of a relatively small number of RNA polymerase molecules from unstable to stable RNA genes along with the increase in the stable RNA chain growth rate is sufficient to account for the abrupt increase in the net rate of RNA synthesis. Furthermore, our calculations indicate that the linear density of polymerase molecules on the ribosomal DNA template increases with the bacterial growth rate, such that in rapidly growing bacteria all ribosomal RNA genes (48 copies at μ = 3) are nearly saturated with RNA polymerase.     

Optimal conditions for the preservation of mouse lymph node cells in liquid nitrogen using cooling rate techniques

The factors that affect the survival of mouse lymphocytes throughout a procedure for storage at ?196 °C have been studied both for the improvement of recovery and the possible extension to the mouse system of cell selection by freezing. After thawing, the survival of cells cooled at different rates in dimethyl sulphoxide (DMSO, 5 or 10%, $\text{v}\text{v}$) was assessed from the [³H]thymidine incorporation in response to phytohaemagglutinin and concanavalin A. Before freezing the protection against freezing damage increased with time (up to 20 min) in DMSO (5%, $\text{v}\text{v}$) at 0 °C. Superimposed upon this effect was toxicity due to the DMSO. During freezing and thawing the cooling rate giving optimal survival was 8 to 15 °C/min for cells in DMSO (5%) and 1 to 3 °C/min for DMSO (10%). Omission of foetal calf serum was detrimental. Rapid thawing (>2.5 °C/min) was superior to slow thawing. After thawing dilution at 25 or 37 °C greatly improved cell survival compared with 0 °C; at 25 °C survival was optimal (75%) at a moderate dilution rate of 2.5 min for a 10-fold dilution in FCS (10%, $\text{v}\text{v}$) followed by gentle centrifugation (50g).Dilution damage during both thawing and post-thaw dilution may be due to osmotic swelling as DMSO and normally excluded solutes leave the cell. The susceptibility of the cell membrane to dilution damage may also be increased during freezing. The need to thaw rapidly and dilute at 25 °C after thawing is probably due to a decrease in dilution stress at higher temperatures. Optimisation of dilution procedures both maximised recovery and also widened the range of cooling rates over which the cells were recovered. These conditions increase the possibility of obtaining good recovery of a mixed cell population using a single cooling procedure. Alternatively, if cell types have different optimal cooling rates, stressful dilution may allow their selection from mixed cell populations.     

The decay of genetic variability in geographically structured populations. II

The ultimate rate of approach to equilibrium in the infinite stepping-stone model is calculated. The analysis is restricted to a single locus in the absence of selection, and every mutant is assumed to be new to the population. Let f(t, x) be the probability that two homologous genes separated by the vector x in generation t are the same allele. It is supposed that $\text{f(}\text{0, x}\text{) = O(x}{}^{\mathrm{?2?\eta }}\text{), η > 0,}\text{as}\text{x ≡ ¦}\text{x}\text{¦ → ∞}$. In the absence of mutation, f(t, x) tends to unity at the rate $\text{t}{}^{\mathrm{<mtext>?1</mtext><mtext>2</mtext>}}$ in one dimension and (ln t)^?1 in two dimensions. Thus, the loss of genetic variability in two dimensions is so slow that evolutionary forces not considered in this model would supervene long before a two-dimensional natural population became completely homogeneous. If the mutation rate, u, is not zero f(t, x) asymptotically approaches equilibrium at the rate $\text{(1 ? u)}{}^{\mathrm{2t}}\text{t}{}^{\mathrm{<mtext>?3</mtext><mtext>2</mtext>}}$ in one dimension and (1 ? u)^2tt^?1(lnt)^?2 in two dimensions. Integral formulas are presented for the spatial dependence of the deviation of f(t, x) from its stationary value as t → ∞, and for large separations this dependence is shown to be (const + x) in one dimension and (const + ln x) in two dimensions. All the results are the same for the Malécot model of a continuously distributed population provided the number of individuals per colony is replaced by the population density. The relatively slow algebraic and logarithmic rates of convergence for the infinite habitat contrast sharply with the exponential one for a finite habitat.     

An x-ray crystallographic study of hemerythrin.

The non-heme iron protein, hemerythrin, has been crystallized from Themiste dyscritum. The crystals belong to the tetragonal space group P4 with unit cell dimensions $\text{a = b = 86.5}\text{A}\text{̊}\text{, c = 80.6}\text{A}\text{̊}$. A 2-fold molecular axis is suggested, implying that the asymmetric unit contains four subunits each with a molecular weight of 12,600.     

Purification and preliminary crystallographic studies on azurin and cytochrome c' from Alcaligenes denitrificans and Alcaligenes sp. NCIB 11015.

Purification and crystallisation procedures are reported for azurin and cytochrome c′ from Alcaligenes denitrificans and Alcaligenes sp. NCIB 11015. The azurin crystals from A. denitrificans are suitable for high-resolution X-ray structure analysis. They are orthorhombic, space group C222₁ (with marked tetragonal pseudo-symmetry), cell dimensions $\text{a = 75.0}\text{A}\text{?}\text{, b = 74.1}\text{A}\text{?}\text{, c = 99.5}\text{A}\text{?}$, with two molecules per asymmetric unit. The cytochrome c′ crystals from both species are hexagonal, space group P6₁22 (or P6₅22), cell dimensions $\text{a = b = 54.7}\text{A}\text{?}\text{, c ~ 185}\text{A}\text{?}$, γ = 120 °, with one subunit (molecular weight 14,000) in the asymmetric unit.     

Crystals of glutamine synthetase from Escherichia coli.

Further details are given of crystals of glutamine synthetase prepared from Escherichia coli. Crystals of two kinds have been observed: (1) rhombic dodecahedra which correspond to the morphology of the crystals studied by Eisenberg et al. (1971) (and which were found by them to contain dodecamers), and (2) rhombohedra, reported here. Cell dimensions and packing considerations led to the consideration of two possible structures for the rhombohedral crystals. These we have called the “T = 7 structure” and the “B.C.C. structure”. The T = 7 structure would be related to that derived by Eisenberg and would contain dodecamers, but is inconsistent with our X-ray intensity data. The B.C.C. structure is considered more probable. It is built of cubic octomers or square tetramers. Electron micrographs of our glutamine synthetase preparations show a wide variety of aggregates, including dodecamers and tetramers. The unit cell dimensions of our crystals are $\text{a = 140 ± 2}\text{A}\text{̊}$, and $\text{c = 148 ± 2}\text{A}\text{̊}$. The Laue symmetry group is $\text{3}\text{̄}$m P3₁.     

Comparative studies between microbiological culture and uptake of uridine/uracil to detect mycoplasmal infection of cell cultures.

Controlled, prospective studies were performed to compare detection of cell culture mycoplasmas by ratio of uptake of tritiated uridine (UdR) to tritiated uracil (U) and by microbiological culture. Culture was by standard agar and broth inoculation with aerobic and anaerobic incubation; immunofluorescent staining of indicator cell cultures was used to detect M. hyorhinis. The ratio of uptake of UdR to U ($\text{UdR}\text{U}$) and interpretation of test results were by standard published methods and performed in triplicate. 115 cell cultures were simultaneously assayed by the two techniques. 84 cultures (73.1%) yielded agreement between the 2 methods; 2 cultures (1.7%) yielded conflicting results, and 29 cultures (25.2%) yielded $\text{UdR}\text{U}$ results in the questionable range. Conflicting results consisted of two negative $\text{UdR}\text{U}$ tests in mouse cell cultures infected with M. orale. In separate studies, 3T-6 cultures freshly infected with M. orale yielded negative $\text{UdR}\text{U}$ results 3 days after infection, questionable results after 10 days and a positive $\text{UdR}\text{U}$ 17 days after infection. $\text{UdR}\text{U}$ detected infection in fibroblast, epithelial, and lymphocyte cell cultures. Highest $\text{UdR}\text{U}$ ratios were detected in human skin fibroblasts at early population doubling levels (PDLs), 4064 in one culture at PDL4. $\text{UdR}\text{U}$ was determined for IMR-90, a human diploid fibroblast at 12 different PDLs using the same lot of media. $\text{UdR}\text{U}$ gradually decreased throughout the life of the culture, from 2 125 at PDL6 to 340 at PDL36. Cultures in phase III and others exhibiting poor growth frequently yielded questionable or false-positive $\text{UdR}\text{U}$ results and were not included in tabulations of these results. $\text{UdR}\text{U}$ determined in endothelial cell cultures decreased as population density increased. In a representative experiment performed over a 4-day period, the $\text{UdR}\text{U}$ values were 1 808, 955 and 356 when the number of endothelial cells in culture were 5.3 × 10⁵, 6.6 × 10⁵ and 1.1 × 10⁶, respectively.     

Crystallography of alpha-lactalbumin. III. Crystals of baboon milk alpha-lactalbumin.

Crystals of baboon α-lactalbumin suitable for structure analysis at high resolution have been obtained. They have cell dimensions $\text{a = 35.5}\text{A}\text{?}\text{, b = 69.1}\text{A}\text{?}\text{, c = 46.1}\text{A}\text{?}$, space group P2₁2₁2 and contain one molecule of α-lactalbumin per asymmetric unit.     

The sites of synthesis and the subsequent migration of newly synthesized protein in retina

Radioautographs of rabbit retinas fixed immediately after a 1 or 2 min exposure in vitro to ³H leucine revealed high rates of protein synthesis in receptor cell inner segments, perikarya of ganglion cells, and cells of the inner nuclear layer. If these brieflly labelled retinas were returned to unlabelled medium for periods of up to 6 hr, the radioautographs revealed a progressive dispersion of the labelled proteins from their sites of synthesis. This was largely completed by $\text{1}\text{1}\text{2}$ hr and appeared, in one instance at least, to involve processes other than simple diffusion. Superimposed on the dispersive phenomenon was a process of concentration of the newly formed proteins at two sites quite distant from their synthesis, that was apparent after $\text{1}\text{1}\text{2}$hr. One of these sites was the receptor cell outer segments, as has been previously described, the other was the outer plexiform layer.     

Preliminary crystallographic data on buffalo beta-lactoglobulin.

Lactoglobulin isolated from Italian buffalo milk has been crystallized in two different forms. Crystals of type I have been grown at pH 3.5 and belong to space group P6₃; the unit cell dimensions are $\text{a = b = 67}\text{A}\text{?}\text{, c = 142}\text{A}\text{?}$, and accordingly the asymmetric unit contains one dimer of molecular weight 36,000. Type II crystals, grown at pH 8.5, have only one monomer per asymmetric unit; the space group is P3₁21 (or enantiomorph) and the unit cell edges are: $\text{a = b = 54.4}\text{A}\text{?}\text{, c = 113.2}\text{A}\text{?}$. The crystals of the trigonal form show low radiation damage and are suitable for a structural investigation.     

Procedure for measurement of amino acid transport from blood to brain in small animals

The exponential plasma specific activity curve 2.5 to 12.5 min after injection (sc) of [¹⁴C]tyrosine was integrated and divided by time to obtain the mathematical relationship between the average equivalent specific activity $\text{S}$ and the measured specific activity S in any individual animal. $\text{S}$ is the constant, average value of S that is equivalent to the curvllinearly varying quantity that the body tissues are actually exposed to. Dividing the total brain radioactivity by $\text{S}$ gave the tissue Tyr uptake U. The function $\text{dU}\text{dt}$ is linear from 2.5 to 12.5 min and represents the rate of uptake of the amino acid. Incorporation into protein was similarly measured. Brain uptake of Tyr averaged 7.06, and the apparent protein incorporation was 1.99 nmol/g of brain per min. The γ-glutamyl cycle inhibitor l-methionine-RS-sulfoximine reduced total brain uptake of tyrosine by 42.8% and the apparent rate of protein incorporation by 39.0%.     

Chemical modification of mitochondria. II. Effect of labeling on oxidative phosphorylation

The labeling of rat liver mitochondria (RLM) by the uncoupler 2,4-dinitro-5-(bromoacetoxyethoxy)phenol (DNBP) was studied and related to the effect of this molecule on oxidative phosphorylation. Alkylation of the cysteine residues was measured both with respect to incubation time of RLM with DNBP and with increasing DNBP concentration. At 3.3 × 10^?5m DNBP, the amount of S-carboxymethyl-cysteine formed was found to level off after about 3 min. The rate of ATP synthesis in RLM is reduced by increasing concentrations of DNBP and falls to zero, with either hydroxybutyrate or succinate as substrate, at 2 × 10^?4m DNBP. To characterize the effect of labeling on oxidative phosphorylation, the $\text{P}\text{O}$ ratio were measured after incubating RLM with DNBP for various times between 10 and 300 sec. The $\text{P}\text{O}$ ratio increases and tends to level off as the incubation time increases. No increase in $\text{P}\text{O}$ ratio was noted when RLM were similarly incubated with the nonlabeling uncoupler 2,4-dinitro-5-(acetoxyethoxy) phenol. Further, the effect of labeling on oxidative phosphorylation was determined with RLM which had been treated with DNBP and then washed free of the excess unreacted uncoupler. DNBP produces specific labeling in RLM which, when related to the effects of this uncoupler on oxidative phosphorylation, suggests that the labeled proteins may be involved in the primary energy transduction process.     

Ordered transcription of RNA tumor virus genomes.

The crystal structure of sodium adenylyl-3′,5′-uridine (ApU) hexahydrate has been determined by X-ray diffraction procedures and refined to an R factor of 0.057. ApU crystallizes with two molecules per asymmetric unit in a monoclinic unit cell, space group P2₁, with cell dimensions: $\text{a = 18.025, b = 17.501, c = 9.677}\text{A}\text{?}\text{and}\text{β = 99.45 °}$. The two independent molecules of ApU form a small segment of right-handed antiparallel double-helical RNA in the crystal, with Watson-Crick base-pairing between adenine and uracil. This is the first time that this Watson-Crick base-pair has been seen unambiguously at atomic resolution and it is also the first time that a nucleic acid fragment with double-helical symmetry has been seen at atomic resolution. The distance between the C1′ atoma of the adenine-uracil base-pair is slightly shorter than the analogous distance seen in guanine-cytosine base-pairs. The bases in each strand are heavily stacked. One sodium cation binds to the phosphates, as expected; however, the other sodium cation binds on the dyad axis in the minor groove of the double helix. It is co-ordinated directly to the two uracil carbonyl groups which protrude into the minor groove and is shielded from the nearest phosphates by a shell of water. This binding appears to be sequence-specific for ApU. One of the adenines also forms a pair of hydrogen bonds to a nearby ribose, utilizing N6 and N7. The 12 water molecules per double-helical fragment are all part of the first co-ordination shell. The ions and the symmetry of the double-helical fragment are the major organizing elements of the solvent region.     

Structural investigations of mode of action of drugs. I. Molecular structure of mitomycin C.

The crystal and molecular structure of the antitumor antibiotic mitomycin C has been determined by X-ray diffraction. The space group is monoclinic P2₁ with cell dimensions $\text{a}\text{=77.988(3)}$, $\text{b}\text{=20.355(7)}$, $\text{c}\text{=9.679(3)}\text{A}\text{?}$, β=95.99°(1) and Z=4. The structure was solved by direct methods. There are two independent molecules per asymmetric unit. The structure was refined to an R value of 0.049 for 2151 observed reflections measured on diffractometer. The benzoquinone ring is slightly deviated from planarity. The N4 in the indole ring behaves like an amide nitrogen owing to its participation in the conjugated benzoquinoid system. The five membered ring through C1, C2, C3, N4 and C9a adopts an envelope conformation. The molecules are held together in crystal by the hydrogen bonds. All nitrogens except N4 are involved in the hydrogen bonding. Studies with models of drug and DNA indicate two different kinds of mechanism for crosslinking.     

Cell growth factor activity: New quantitative method in cell culture assay

This quantitative method for the assay of growth factor activity in vitro was developed empirically. Based on the empirical equations, it seems logical to express the cell growth rate by cell duplication frequency, f, and the growth factor activity by the normalized cell growth rate: $\text{1}\text{(T-T}\text{min}\text{)}$. The latter is recommended as the basis of defining an arbitrary unit of growth factor activity. For the in vitro growth factor activity assay, the quantitative method based on the empirical equations has two important features: (1) it appears to express linearly the growth factor concentration in assay, and (2) it is not affected by the initial cell counts and the length of cultivation.The view that these two features overcome the apparent defects associated with the expression systems that use the cell count ratio and net cell count is discussed. The quantitative method based on the empirical equations has been verified with experiments and compared with expression systems using the cell count ratio and net cell count. The experiments, which verified the quantitative method based on the empirical equations, were carried out with four selected cell lines: aortic endothelial cells, mouse 3T3 fibroblasts, Walker 256 carcinoma, and Chang's liver cells, using serum as the source of growth factor(s) for all cell lines. Vitreous, retinal extract, and Walker 256 carcinoma extract were also used for aortic endothelial cells.     

Preliminary crystallographic study of omega-amino acid: pyruvate aminotransferase from Pseudomonas sp. F-126.

Large single crystals of ω-amino acid: pyruvate aminotransferase, were prepared by dialysis of the enzyme solution against 2.2 m-ammonium sulphate solution at pH 7.8. X-ray diffraction patterns show that the crystals belong to the orthorhombic space group I222 or I2₁2₁2₁ with unit cell dimensions $\text{a = 124.1}\text{A}\text{?}\text{, b = 137.9}\text{A}\text{?}\text{, and}\text{c = 61.2}\text{A}\text{?}$. The asymmetric unit consists of one monomer of molecular weight 43,000.     

Crystallization of cow α-lactalbumin

Three new crystal forms of cow α-lactalbumin are described. A trigonal form in space group P3₁21 or P3₂21 has unit cell dimensions: $\text{a = b = 57.4}\text{A}\text{?}\text{, c = 75.0}\text{A}\text{?}$. A hexagonal form in space group P622 has unit cell dimensions: $\text{a = b = 94.0}\text{A}\text{?}\text{, c = 67.1}\text{A}\text{?}$. A second trigonal form grown in the presence of calcium ions belongs to space group P321 with unit cell dimensions: $\text{a = b = 93.7}\text{A}\text{?}\text{, c = 66.9}\text{A}\text{?}$. The significance of these new crystal forms to the structure determination of cow α-laetalbumin is discussed.