Construction and properties of a recombinant plasmid containing gene 32 of bacteriophage T4D

This paper describes the construction and characterization of a chimeric plasmid that encodes the single-stranded DNA-binding protein of bacteriophage T4D (the product of gene 32). The plasmid contains a 2·6 × 10³ base HindIII segment of T4 DNA that includes genes 59 and 32 as well as a portion of gene 33. Isolation of bacteria carrying the recombinant plasmid became possible when the segment of phage DNA contained an amber mutation in gene 32. This suggests that a functional gene 32 is deleterious to the cell. Using antibody to gene 32 protein, we have been able to demonstrate expression of the plasmid-borne gene 32 in uninfected bacteria. Deletion variants of the gene 32 plasmid have been constructed in vitro. These have been used to align the genetic map of the region with the restriction map and to study phage gene expression from the plasmid in both infected and uninfected cells. In phage-infected cells the level of functional gene 32 product regulates the efficiency of translation of its own messenger RNA. We also observe such self-regulation for gene 32 present on the plasmid.     

Histrionicotoxin and alkylguanidine interactions with the solubilized and membrane-bound muscarinic acetylcholine receptor from porcine atria

The interaction of alkylguanidines and decahydrohistrionicotoxin with the membrane-bound and solubilized muscarinic acetylcholine receptor (mAcChR) from porcine atria was described. Alkylguanidines with alkyl chain lengths from one to ten carbons displaced l-[³H]quinuclidinyl benzilate (l-[³H]QNB) competitively from a single class of sites for the membrane-bound mAcChR. From a plot of ?ln K_i versus alkyl carbon chain number, a value of ?(473 ± 30) cal/mol was estimated as the energetic contribution per methylene group to the total binding energy. The binding of alkylguanidines to the digitonin/cholate solubilized mAcChR was complex in nature resulting in titration curves that did not obey the law of mass action for simple competitive inhibition at higher alkyl carbon numbers and a sigmoidal plot of ?ln K_i versus carbon number. Decahydrohistrionicotoxin bound in a competitive manner versus l-[³H]QNB to both the membrane-bound (K_i = (6.9 ± 1.4) × 10^?6 M) and the solubilized (K_i = (1.5 ± 0.3) × 10^?5 M) preparations.     

Study of the incompatibility and replication of the 70-kb virulence plasmid of Yersinia

The 70-kb virulence plasmid, vir, from four Yersinia enterocolitica and one Y. pseudotuberculosis strains are incompatible with IncFI plasmids FLac and R386 while they are compatible with plasmids representing nine other incompatibility groups. Hybridization experiments carried out on one of these virulence plasmids showed that it contains the F incompatibility determinant D, incD. This determinant was cloned onto pACYC184 and the recombinant clone expressed incompatibility with FLac. We conclude that the incompatibility observed between F or R386 and the 70-kb virulence plasmid of Y. enterocolitica and Y. pseudotuberculosis is mediated by incD. Replication genes (rep) from the same plasmid were cloned independently in Escherichia coli. Rep and incD map on two different BamHI fragments. Surprisingly, the replicon isolated is not sensitive to inc D incompatibility. Apart from incD, vir and F share extremely little homology. In particular, there is no evidence for the presence of an F-like transfer operon on vir.     

Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system

The relative quantities of 26 known transfer RNAs of Escherichia coli have been measured previously (Ikemura, 1981). Based on this relative abundance, the usage of cognate codons in E. coli genes as well as in transposon and coliphage genes was examined. A strong positive correlation between tRNA content and the occurrence of respective codons was found for most E. coli genes that had been sequenced, although the correlation was less significant for transposon and phage genes. The dependence of the usage of isoaccepting tRNA, in E. coli genes encoding abundant proteins, on tRNA content was especially noticeable and was greater than that expected from the proportional relationship between the two variables, i.e. these genes selectively use codons corresponding to major tRNAs but almost completely avoid using codons of minor tRNAs. Therefore, codon choice in E. coli genes was considered to be largely constrained by tRNA availability and possibly by translational efficiency. Based on the content of isoaccepting tRNA and the nature of codon-anticodon interaction, it was then possible to predict for most amino acids the order of preference among synonymous codons. The synonymous codon predicted in this way to be the most preferred codon was thought to be optimized for the E. coli translational system and designated as the “Optimal codon”. E. coli genes encoding abundant protein species use the optimal codons selectively, and other E. coli genes, such as amino acid synthesizing genes, use optimal and “non-optimal” codons to a roughly equal degree. The finding that the frequency of usage of optimal codons is closely correlated with the production levels of individual genes was discussed from an evolutionary viewpoint.     

Regions on the F plasmid which affect plasmid maintenance and the ability to segregate into Escherichia coli minicells

Certain derivative mini-F plasmids were found to segregate into Escherichia coli minicells, in contrast to the intact mini-F plasmid which does not. Segregation was not related to the presence or absence of the normal origin of vegetative replication, but appeared to be affected by regions of F which encode replication, incompatibility, copy number control, and partitioning functions. Segregation of mini-F plasmids into minicells was not random; the plasmid concentration in minicells did not correlate with the plasmid concentration in cells. Genes, or gene products, of F from the region spanning the sequences 44.1–49.3F appeared to affect the ability of mini-F plasmids to segregate into minicells. Segregation of mini-F plasmids into minicells was not directly related to stable plasmid inheritance. These results argue for the sequestration of mini-F plasmids in host cells.     

Molecular characterization of a stable Flac plasmid

F$\text{lac}$S is a thermostable extrachromosomal element isolated in $\text{Salmonella typhimurium}$ which is altered in its replication as compared to its precursor F_ts114$\text{lac}$. Sedimentation of both these plasmids in alkaline sucrose gradients has indicated a difference in their sizes. Contour length measurements of open circular plasmid DNA molecules photographed in the electron microscope have revealed the estimated molecular weight of F_ts114$\text{lac}$ to be 81 × 10⁶ daltons while that of F$\text{lac}$S is 109 × 10⁶ daltons. F$\text{lac}$S may carry a segment of $\text{S. typhimurium}$ chromosomal or cryptic plasmid DNA.     

Sodium periodate stimulation of human lymphocytes : A comparison between normal and chronic lymphocytic leukemia lymphocytes

Lymphocytes from healthy donors and from patients with chronic lymphocytic leukemia (CLL) were stimulated to divide with sodium periodate. The time of maximal response of normal lymphocytes to sodium periodate (NaIO₄) was earlier than that observed to phytohemagglutinin (PHA), but the magnitude was lower. In comparison, CLL lymphocytes responded to NaIO₄ more extensively and earlier than to PHA.     

Choline sulfokinase of Penicillium chrysogenum: partial purification and kinetic mechanism.

Choline sulfokinase (3′-phosphoadenosine 5′-phosphosulfate (PAPS):choline sulfotransferase, EC 2.8.2.6) was purified approximately 30-fold from the mycelium of Penicillium chrysogenum. The K_m for PAPS is 12 μm. The enzyme is remarkably specific for the adenosine 3′,5′ (or 2′-5′)-diphosphate moiety. 3′,5′-ADP (PAP) has a K_i of 2.5 to 14 μm (depending on the choline concentration) whereas the K_i values of 3′-AMP, 5′-AMP, and 5′-ADP are at least 300-fold higher. The enzyme is also highly specific for choline (K_m = 17 μM). Of a number of other amino alcohols tested, none were potent inhibitors and only dimethylaminoethanol served as a reasonably good substrate (K_m = 800 μmV = 35% of V with choline). Triethylaminoethanol was a significantly poorer substrate (K_m = 2800 μM; V = 2% of V with choline). The purified enzyme is relatively stable when stored frozen in the presence of 25% sucrose. In the absence of sucrose, the maximum activity decreases and the K_m for choline increases. (The K_m for PAPS remains constant.) The age-inactivated enzyme can be restored to full activity (original V and K_m for choline) by a 10-min preincubation with 50 mm mercaptoethanol. However, prolonged incubation (24 h) with 50 mm mercaptoethanol results in irreversible denaturation. Initial velocity studies established that the enzyme follows a sequential kinetic mechanism. Product inhibition studies suggest a rapid equilibrium random binding sequence. Choline-O-phosphate (a dead-end inhibitor) is linearly competitive with choline and a linear mixed type inhibitor with respect to PAPS. Choline analogs lacking the alcohol (or ester) group (e.g., trimethylammonium, neurine, chlorocholine) are competitive dead-end inhibitors with respect to choline but are uncompetitive with respect to PAPS. Thiocholine is a linear mixed type inhibitor with respect to PAPS, but the reciprocal plots are almost parallel. These results suggest that the analogs lacking an oxygen atom have a negligible affinity for the free enzyme and bind predominantly to the enzyme-PAPS complex.     

Sensitive detection of specific repair endonucleases: radial diffusion assay utilizing differential alkaline denaturation of supercoiled and nicked PM2 DNA in agarose gels

An improved method for separation and quantitation of sulfated neutral and acidic steroids in human feces was developed. The procedure consists of separation of sulfated steroids on Sephadex LH-20 and hydrolysis by cholylglycine hydrolase followed by quantitation and identification of the trimethylsilylether derivatives by gas-liquid chromatography and gas-liquid chromatography-mass spectroscopy. Using this procedure, we detected no sulfated bile acids in human feces. However, sulfated cholesterol was detected in the sulfated bile acid fraction obtained from human fecal extracts. Analysis showed that cholesterol sulfate comprised 12.3, 11.2, and 31.0% of the total neutral sterol fraction in the three fecal samples. Using our procedures, cholesterol sulfate and bile acid sulfates in a biological mixture can be quantitated and identified when they are present.     

Tn5 insertions in the agrocin 84 plasmid: the conjugal nature of pAgK84 and the locations of determinants for transfer and agrocin 84 production

The kanamycin-resistance transposon Tn5 was randomly introduced into pAgK84, a 47.7-kb plasmid coding for agrocin 84 production in Agrobacterium. Using such marked plasmids, pAgK84 was found to be conjugal. It could be transferred to several Agrobacterium strains including those harboring octopine- or nopaline-type Ti plasmids. Its presence has no effect on Ti plasmid functions such as opine utilization and tumorigenicity, but it does confer agrocin 84 immunity upon previously sensitive strains. The plasmid could also be conjugally transferred to a Nod+ Fix+ strain of Rhizobium meliloti. The production of agrocin 84 is expressed in all Agrobacterium and Rhizobium transconjugants tested. The agrocin plasmid could not be introduced into restrictionless Escherichia coli or Pseudomonas aeruginosa recipients by conjugation or transformation. The sites of 92 independent Tn5 insertions were mapped on pAgK84. These insertions are dispersed over the entire length of the plasmid. Analysis of the sites and effects of the Tn5 insertions has allowed us to construct a functional map of pAgK84. Forty-three of these insertions, spanning a 20-kb segment of the plasmid, abolished or greatly reduced the production of agrocin 84. The presence of two insertions within this segment having an effect on agrocin production suggests that at least three regions of the plasmid are involved in agrocin 84 biosynthesis. Fourteen of the Tn5 insertion derivatives are no longer conjugally transferable. These insertions all map to a single region of the plasmid and define about 3.5-kb as being associated with transfer functions.     

The chemistry of thujone: I. Synthesis of insect juvenile hormone analogs via wittig coupling

Syntheses of the C₈ and C₁₀ olefinic units cis- and trans-5-ethyl-1-iodo-hex-4-enes and cis- and trans-7-ethyl-3-iodo-oct-6-enes are described. The Wittig coupling of such units with derivatives of α- and β-thujaketonic acids to give analogs of insect juvenile hormones is discussed.     

Nutritional and hormonal control of lung and liver fatty acid synthesis.

During starvation and in streptozotocin-induced diabetes, the total activities of rat lung acetyl CoA carboxylase and fatty acid synthetase are reduced to one-third of the normal values. Refeeding of the starved animals or administration of insulin to diabetic animals restores the levels to the original values. The insulin effect is dose and time dependent. These data contrast with those in the liver, where a 30- to 50-fold depression of these enzymes is observed in the diabetic state and administration of insulin is actually followed by doubling of the activity over normal controls. Fat-free high-fructose diet (containing 60% fructose by weight) enhances the activities of liver enzymes 3- to 6-fold over the values of controls on laboratory diet but has no effect on the lung enzymes. Long-term feeding of fructose diet also increases the activities of liver enzymes from diabetic animals to twice the value of normal controls on laboratory diet. Insulin administration to fructose-fed diabetic animals restores the enzyme activities to those obtained with fructose-fed normal controls. However, the stimulation of lung enzymes of diabetic animals can be effected either by fructose or by insulin. Antigen-antibody titrations and measurements of the rate of protein synthesis show that the increased activity of the lung and liver fatty acid synthetase is due to enhanced content rather than increased specific activity. These data suggest that insulin or fructose effects on fatty acid-synthesizing enzymes are mediated through intermediate(s) whose concentration is affected in the experimental diabetes. Furthermore, all tissues may not have stringent insulin requirements since the lung enzymes can be stimulated by fructose alone.     

Identification and mass analysis of human fibrinogen molecules and their domains by scanning transmission electron microscopy

The domainal substructure and molecular conformation of human fibrinogen have been investigated by evaluating scanning transmission electron microscopic images of freeze-dried or negatively contrasted native fibrinogen (fractions I-4 and I-9), glutaraldehyde-treated fibrinogen, or plasmic core fragments D₁ and E₂. Although some unstained freeze-dried native or glutaraldehyde-treated fibrinogen molecules were relatively compact and even occasionally spheroidal, typical images were elongated symmetrical tridomainal structures 460 Å ± 20 Å in length; frequently they were bent into a variety of elongated though non-linear arrangements. Their identification as monomolecular forms of fibrinogen by scanning transmission electron microscopic mass measurements resolved uncertainties relating to the identity of such objects as single molecules. The central domains of fraction I-4 molecules had a greater mass than those of fraction I-9 (1.01 × 10⁵M_rversus 7.5 × 10 M_r, respectively). This difference accounted for the observed mass difference between fraction I-4 and fraction I-9 molecules (i.e. 3.27 × 10⁵M_rversus 2.97 × 10⁵M_r, respectively) and suggested that the COOH-terminal region of the Aα chain (major portions of which are always absent from fraction I-9 molecules) is situated within the mass integration radius for the central domain. When the COOH-terminal region of the Aα chain was present it appeared in negative stain as a thread-like structure originating between the middle and outer domains and extending toward the central domain, sometimes appearing to wind around the long axis.The outer domains of negatively stained molecules resembled negatively stained images of fragment D₁ and could frequently be resolved into at least two discrete subdomains, forming an oblong structure usually canted at an angle of ～120 ° to 150 ° relative to the long axis. Our findings are consistent with prevailing tridomainal structural models of fibrinogen and suggest that these molecules are flexible and may exist in unfolded configurations, or as relatively compact, partially or completely folded forms.     

Structural dynamics of bacterial ribosomes: V. Magnesium-dependent dissociation of tight couples into subunits: Measurements of dissociation constants and exchange rates

The magnesium ion-dependent equilibrium of vacant ribosome couples with their subunits

$\text{70}\text{S}\text{?}\text{k}{}_{\mathrm{?1}}\text{k}{}_1\text{50}\text{S}\text{+30}\text{S}$

has been studied quantitatively with a novel equilibrium displacement labeling method which is more sensitive and precise than light-scattering. At a concentration of 10^?7m, tight couples (ribosomes most active in protein synthesis) dissociate between 1 and 3 mm-Mg²⁺ at 37 °C with a 50% point at 1.9 mm. The corresponding association constants K_a′ are 5.1 × 10⁵m^?1 (1 mm-Mg²⁺), 3.5 × 10⁷m^?1 (2 mm), and 1.2 × 10⁹m^?1 (3 mm), about five orders of magnitude higher than the K_a′ value of loose couples studied by Spirin et al. (1971) and Zitomer & Flaks (1972).In this range of Mg²⁺ concentrations ($\text{37 °C, 50 mm-}\text{NH}{}_4{}^{\mathrm{+}}$) the rate constants depend exponentially and in opposite ways on the Mg²⁺ concentration: k₁ = 2.2 × 10^?3s^?1, k_?1 = 7.7 × 10⁴m^?1s^?1 (2mm-Mg²⁺); k₁ = 1.5 × 10^?4s^?1, k_?1 = 1.7 × 10⁷m^?1s^?1 (5 mm-Mg²⁺). Under physiological conditions (Mg²⁺ ～- 4 mm, ribosome concn ～- 10^?7m), the equilibrium strongly favors association and the rate of exchange is slow ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～- 10}\text{min}$). In the range of dissociation (2 mm-Mg²⁺), association of subunits proceeds without measurable entropy change and hence ΔG^O = ΔH^O. The negative enthalpy change of ΔH^O = ? 10 kcal suggests that association of subunits involves a shape change.Below a critical Mg²⁺ concentration (～- 2 mm), the 50 S subunits are converted irreversibly into the b-form responsible for the transition to loose couples. The results are compatible with two classes of binding sites, one class binding Mg²⁺ non-co-operatively and contributing to the free energy of association by reduction of electrostatic repulsion, and another class probably consisting of hydrogen bonds between components at opposite interfaces whose critical spatial alignment rapidly denatures in the absence of stabilizing magnesium ions.     

Positions of proteins S6, S11 and S15 in the 30 S ribosomal subunit of Escherichia coli

A map of the positions of 12 of the 21 proteins of the 30 S ribosomal subunit of Escherichia coli (S1, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12 and S15), based on neutron scattering, is presented and discussed. Estimates for the radii of gyration of these proteins in situ are also obtained. It appears that many ribosomal proteins have compact configurations in the particle.     

Mechanisms of the multiphasic kinetics in the folding and unfolding of globular proteins

The multiphasic kinetics of the protein folding and unfolding processes are examined for a “cluster model” with only two thermodynamically stable macroscopic states, native (N) and denatured (D), which are essentially distributions of microscopic states. The simplest kinetic schemes consistent with the model are: N-(fast) → I-(slow) → D for unfolding and N ← (fast)-D₂ ← (slow)-D₁ for refolding. The fast phase during the unfolding process can be visualized as the redistribution of the native population N to I within its free energy valley. Then, this population crosses over the free energy barrier to the denatured state D in the slow phase. Therefore, the macrostate I is a kinetic intermediate which is not stable at equilibrium. For the refolding process, the initial equilibrium distribution of the denatured state D appears to be separated into D₁ and D₂ in the final condition because of the change in position of the free energy barrier. The fast refolding species D₂ is due to the “leak” from the broadly distributed D state, while the rest is the slow refolding species D₁, which must overpass the free energy barrier to reach N. At an early stage of the folding process the amino acid chain is considered to be composed of several locally ordered regions, which we call clusters, connected by random coil chain parts. Thus, the denatured state contains different sizes and distributions of clusters depending on the external condition. A later stage of the folding process is the association of smaller clusters. The native state is expressed by a maximum-size cluster with possible fluctuation sites reflecting this association. A general discussion is given of the correlation between the kinetics and thermodynamics of proteins from the overall shape of the free energy function. The cluster model provides a conceptual link between the folding kinetics and the structural patterns of globular proteins derived from the X-ray crystallographic data.