Reverse genetic analysis of the glutathione metabolic pathway suggests a novel role of<Emphasis Type="Italic"> PHGPX</Emphasis> and<Emphasis Type="Italic"> URE2</Emphasis> genes in aluminum resistance in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

We have taken a systematic genetic approach to study the potential role of glutathione metabolism in aluminum (Al) toxicity and resistance, using disruption mutants available in Saccharomyces cerevisiae. Yeast disruption mutants defective in phospholipid hydroperoxide glutathione peroxidases (PHGPX; phgpx1 , phgpx2 , and phgpx3), were tested for their sensitivity to Al. The triple mutant, phgpx1 /2/3, was more sensitive to Al (55% reduction in growth at 300 M Al) than any single phgpx mutant, indicating that the PHGPX genes may collectively contribute to Al resistance. The hypersensitivity of phgpx3 to Al was overcome by complementation with PHGPX3, and all PHGPX genes showed increased expression in response to Al in the wild-type strain (YPH250), with maximum induction of approximately 2.5-fold for PHGPX3. Both phgpx3 and phgpx1/2/3 mutants were sensitive to oxidative stress (exposure to H₂O₂ or diamide). Lipid peroxidation was also increased in the phgpx1/2/3 mutant compared to the parental strain. Disruption mutants defective in genes for glutathione S-transferases (GSTs) (gtt1 and gtt2), glutathione biosynthesis (gsh1 and gsh2), glutathione reductase (glr1) and a glutathione transporter (opt1) did not show hypersensitivity to Al relative to the parental strain BY4741. Interestingly, a strain deleted for URE2, a gene which encodes a prion precursor with homology to GSTs, also showed hypersensitivity to Al. The hypersensitivity of the ure2 mutant could be overcome by complementation with URE2. Expression of URE2 in the parental strain increased approximately 2-fold in response to exposure to 100 M Al. Intracellular oxidation levels in the ure2 mutant showed a 2-fold (non-stressed) and 3-fold (when exposed-to 2 mM H₂O₂) increase compared to BY4741; however, the ure2 mutant showed no change in lipid peroxidation compared to the control. The phgpx1/2/3 and ure2 mutants both showed increased accumulation of Al. These findings suggest the involvement of PHGPX genes and a novel role of URE2 in Al toxicity/resistance in S. cerevisiae.Communicated by D.Y. Thomas     

The ability of acidic pH,growth inhibitors,and glucose to increase the proton motive force and energy spilling of amino acid-fermenting <Emphasis Type="Italic">Clostridium sporogenes</Emphasis> MD1 cultures

Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (p) was only –25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZpH) and the electrical membrane potential () increased. The p was –125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but increased to –70 mV. Protein synthesis inhibitors also significantly increased . Non-growing, arginine-energized cells had a of –80 mV at pH 6.7 or pH 4.7, but was not detected if the F₁F₀ ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large p to grow, and p appears to serve as a mechanism of ATP dissipation or energy spilling.Mandatory disclaimer: Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.     

Sodium ions are necessary for growth and energy transduction in the marine cyanobacterium Oscillatoria brevis

The maximal growth rate of the marine cyanobacterium Oscillatoria brevis was reached at 200–400 mM NaCl and pH 9.0–9.6. NaCl was found (i) to stimulate the rate of the light-supported generation across the cytoplasmic membrane of the cells and (ii) to decrease the sensitivity of level and motility of the O. brevis trichomes to protonophorous uncouplers. The Na⁺/H⁺ antiporter, monensin, increased both and the uncoupler sensitivity of the cells. The data obtained agree with the assumption that O. brevis possesses a primary Na⁺ pump in its cytoplasmic membrane.Abbreviations ATP adenosine-5-triphosphate - TTFB tetrachlortrifluoromethylimidazol - CCCP carbonyl cyanide m-chlorophenylhydrazone - Na⁺ transmembrane electrochemical potential differences of Na⁺ - transmembrane electric potential difference - pNa transmembrane pNa difference     

Genetic analysis of carbon isotope discrimination and agronomic characters in a bread wheat cross

Carbon isotope discrimination () has been suggested as a selection criterion to improve transpiration efficiency (W) in bread wheat (Triticum aestivum L.). Cultivars Chinese Spring with low A (high W) and Yecora Rojo with high (low W) were crossed to develop F₁, F₂, BC₁, and BC₂ populations for genetic analysis of and other agronomic characters under well-watered (wet) and water-stressed (dry) field conditions. Significant variation was observed among the generations for only under the wet environment. Generation x irrigation interactions were not significant for . Generation means analysis indicated that additive gene action is of primary importance in the expression of under nonstress conditions. Dominance gene action was also detected for , and the direction of dominance was toward higher values of . The broad-sense and the narrow-sense heritabilities for were 61 % and 57% under the wet conditions, but were 48% and 12% under the draughted conditions, respectively. The narrow-sense heritabilities for grain yield, above-ground dry matter, and harvest index were 36%, 39%, and 60% under the wet conditions and 21%, 44%, and 20% under dry conditions, respectively. The significant additive genetic variation and moderate estimate of the narrow-sense heritability observed for indicated that selection under wet environments should be effective in changing in spring bread wheat.     

Kinetics of the flash-induced electrochromic absorbance change in the presence of background illumination. Turnover rate of the electron transport. I. Isolated intact chloroplasts

We investigated the flash-induced electrochromic absorbance change, A ₅₁₅, of isolated intact chloroplasts in continuous monochromatic background light of different intensities and wavelengths. From the variation of the amplitude of A ₅₁₅ in background illumination the steady-state turnover time of electron transport was found to be around 100 msec and the slowest process could be assigned to a photosystem 1 driven cycle. The change of pH induced by nigericin, ATP, or ADP did not modify substantially the turnover time.In contrast to earlier observations the slow rise (10 msec) of A ₅₁₅ in untreated chloroplasts persists also at high-intensity background illumination exciting both photosystems. The proportion of the slow rise of A ₅₁₅ in nigericin-treated chloroplasts increases in the presence of background light. This result on the slow rise is discussed in terms of two different models existing in the literature.     

Mechanisms of voltage transients during current clamp inNecturus gallbladder

Summary Microelectrode techniques were employed to study the mechanisms of the transepithelial voltage transients (V _ms ) observed during transmural current clamps in the isolatedNecturus gallbladder. The results indicate that: a) part of V _ms is due to a transepithelial resistance change (R _t ), and part to a tissue emf change. b) R _t is entirely caused by changes of the resistance of the paracellular pathway. At all current densities employed, the measured changes are probably due to changes in both fluid conductivity and width of the lateral intercellular spaces. At high currents, in addition to the effects on the lateral spaces, the resistance of other elements of the pathway (probably the limiting junction) drops, regardless of the direction of the current. c) The magnitude and polarity of the R _t -independent transepithelial and cell membrane potential transients indicate that the largest emf change takes place at the basolateral membrane (E _b ), with smaller changes at the luminal membrane (E _a ) and the paracellular (shunt) pathway (E _s ). It is shown that two-thirds of the transient are caused by E _s , and one-third by (E _b –E _a ). E _s can be explained by a diffusion potential generated by a current-dependent NaCl concentration gradient across the tissue. E _a and E _b are caused by [K] changes, mainly at the unstirred layer in contact with the basolateral membrane.     

Interaction of cytochrome c L with methanol dehydrogenase from Methylophaga marina 42:

The reaction of methanol dehydrogenase with cytochrome c _L from Methylophaga marina and the reactions of the non-physiological substrates, Wurster's blue and ascorbic acid, with both proteins were studied as a function of temperature (4–32 °C), pressure (1–2000 bar) and ionic strength using the optical high pressure stopped-flow method. The thermodynamic parameters H, S and V were determined for all reactions where electron transfers are involved. These data allowed the determination of the Maxwell relationships which proved the internal thermodynamic consistency of the system under study. A conformational change on the cytochrome c _L level was deduced from both breaks in the Arrhenius plots and the variation of the V with temperature.Abbreviations MOPS 4-morpholinepropanesulfonic acid - CHES 2-(cyclohexylamino)ethanesulfonic acid - MDH methanol dehydrogenase - EDTA ethylenedinitrilotetraacetic acid disodium salt - BTB bromothymol blue (3,3-dibromothymolsulfoneph-thalein) - PQQ 2,7,9-tricarboxy-lH-pyrrolo-[2,3f]quinoline-4,5-dione - cytochrome c _HH mammalian horse heart cytochrome c     

Basis of the Relationship between Ash Content in the Flag Leaf and Carbon Isotope Discrimination in Kernels of Durum Wheat

The relationship between ash content and carbon isotope discrimination () was studied in durum wheat (Triticum durum Desf.) grown in a Mediterranean region (Northwest Syria) under three different water regimes (hereafter referred to as environments). In two of these environments, 144 genotypes were cultivated under rain-fed conditions. In the third environment, 125 genotypes were cultivated under irrigation. Ash content was measured in the flag leaf about 3 weeks after anthesis, whereas was analysed in mature kernels. Total transpiration of the photosynthetic tissues of the culm contributing, from heading to maturity, to the filling of kernels was also estimated. Leaf ash content, expressed either on dry matter or leaf area basis or as total ash per blade, correlated positively (p< 0.001) with in the three environments. However, this relationship was not the result of a positive correlation across genotypes between and tissue water content. Moreover, only a small part of the variation in across genotypes was explained by concomitant changes in ash content. When all genotypes across the three environments were plotted, and ash content followed a non-linear relationship (r ² = 74), with tending to a plateau as the ash content increased. However, for the set of genotypes and environments combined, total ash content per leaf blade was positively and linearly related (r ² = 0.76) with the accumulated culm transpiration. The non-linear nature of the relationship between ash content and is sustained by the fact that culm transpiration also showed a non-linear relationship with kernel . Therefore, differences in leaf ash content between environments, and to a lesser extent between genotypes, seem to be brought about by variations in accumulated transpiration during grain formation.     

Positional specificity in the incorporation of isomeric cis- and trans-octadecenoic acids into glycerolipids of cultured soya cells

Heterotrophically grown cell suspension cultures of soya (Glycine max L.) were incubated with two different mixed substrates consisting of positional isomers of either cis-[1-¹⁴C]octadecenoic acids (8 to 15) or trans-[1-¹⁴C]octadecenoic acids (8 to 16), each with known composition. With both substrates, about one-fourth of the radioactivity supplied was incorporated into the diacylglycerophosphocholines, while another one-fourth of the radioactivity was almost equally distributed between diacylglycerophos-phoethanolamines and triacylglycerols. All the positional isomers of cis-and trans-octadecenoic acids supplied to the cells were readily incorporated into various classes of glycerolipids. None of the octadecenoic acids was isomerized, elongated or desaturated during incubation. From the cis-octadecenoic acids, only the naturally occurring 9-isomer (oleic acid) was preferentially incorporated into position 2 of diacylglycerophosphocholines, diacylglycerophospho-ethanolamines, and triacyglycerols; all the other isomers exhibited a strong affinity for position 1 of the glycerophospholipids and positions 1 and 3 of the triacylglycerols. From the trans-octadecenoic acids, only the 9-isomer (elaidic acid) was preferentially incorporated into position 2 of diacylglycerophospho-cholines and triacylglycerols; all the other isomers preferred position 1 and positions 1 and 3, respectively, of these lipids. In diacylglycerophospho-ethanolamines, however, each of the trans-octadecenoic acids, including the 9-isomer, exhibited a strong affinity for position 1. Apparently, the enzymes involved in the incorporation of exogenous monounsaturated fatty acids into membrane lipids of plant cells can recognize the preferred substrate in a mixture of closely related isomers.     

Effect of different carbon sources on the biosynthesis of polyunsaturated fatty acids ofα-linolenic acid family in culture of minimal deviation hepatoma 7288 c cells

Summary The effect of three different carbon sources on the biosynthesis of polyunsaturated fatty acids of the-linolenic acid series was investigated in hepatoma tissue culture (HTC) cells. Alpha linolenic acid was converted to higher homologs by a desaturating route that synthetized mainly 18:4 (6, 9, 12, 15), 20:4 (8, 11, 14, 17) and 20:5 (5, 8, 11, 14, 17) and an elongating route that produced 20:3 (11, 14, 17) and 20:4 (5, 11, 14, 17) acids. Fasting decreased both biosynthetic routes whereas glucose reactivated only the elongating pathway. Lactalbumin hydrolysate enhanced significantly only the desaturating route whereas glycerol was inactive. Glucose and aminoacids increased similarly the incorporation of labeled linolenic acid in the cells. The results are independent of hormonal effects.Members of the Carrera del Investigador Científico of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.     

A deletion derivative of the kalilo senescence plasmid forms hairpin and duplex DNA structures in the mitochondria of Neurospora.

Summary A novel deletion derivative, kal, of the kalilo senescence plasmid from Neurospora intermedia, was recovered from a culture treated with chloramphenicol. The deletion derivative exists in mitochondria as two different, equally abundant forms: a 2.8 kb duplex DNA molecule kal-2.8) and a 1.4 kb hairpin form kal-1.4). The kal-2.8 plasmid contains the 1366 by terminal inverted repeats and a partially duplicated 102 by segment of the unique sequence of the 8.6 kb kalilo plasmid. In contrast, the kal-1.4 hairpin plasmid appears to result from the folding of single strands that are generated during the replication of kal-2.8. Both forms of kal have covalently linked terminal proteins. Sequence analysis suggests that kal was generated either by slippage of the tip of a growing strand during the replication of kalilo, or by illegitimate recombination between two copies of the plasmid at non-homologous palindromic sequences that might form cruciform structures. In either case, the deletion process was mediated at least in part by an inverted repeat of 5 by in the unique region of kalilo. Since the terminal segments of kalilo DNA that are implicated in plasmid integration might also form cruciform structures, it is possible, but improbable, that the process that generated the first kal molecule is related to that which mediates integration of the plasmid into mitochondrial DNA.     

The neural basis of a sensory filter in the Jamming Avoidance Response: No grandmother cells in sight

Summary The Jamming Avoidance Response (JAR), during which weakly electric fish modulate their electric organ discharge rate in response to a foreign electric signal of nearly the same frequency is strongest for frequency differences (f s) between 3–8 Hz. We have searched for neural correlates of this behavioral specificity. Single unit recordings in the anterior lateral line ganglion (ALLG), the posterior lateral line lobe (PLLL) and the torus semicircularis (TS) ofEigenmannia virescens were made during electrical stimulation simulating jamming by a nearby conspecific.Contrary to previously published reports (Scheich 1974, 1977) we conclude that f specificity does not lie in a single class of receptors or higher-order units in the PLLL tuned to the most effective f s. No tuning is seen at the receptor level of the PLLL. Specificity seems to be a population effect first visible at the level of the torus semicircularis, with individual units responding most strongly to different f s, but with most units tuned to approximately + and-4 Hz. By having cells tuned to a variety of f s but occurring in proportions corresponding to the observed behavior (and the degree to which f s impair electrolocation), animals would be better equipped to carry out other tasks such as detection of relative motion of objects in space and would also be better able to read complex stimuli corresponding to the more usual case of simultaneous jamming from several conspecifics (Partridge and Heiligenberg 1980).Units in the PLLL show slight differences in the timing of their firing to jamming signals presented at a frequency slightly above (+f) the fish's pacemaker frequency compared to those presented at a frequency slightly below (–f) (Scheich 1977). Firing pattern within the beat cycle produced by interaction of the fish's EOD, or an electrical mimic, S₁, and the foreign signal, S₂, is largely unaffected by the field orientation of the jamming signal. In the torus, by contrast, two classes of units are encountered which completely reverse the pattern of their firing within the beat cycle if the sign of the f is reversed. And, unlike the PLLL cells, those in TS respond differentially to different stimulus field geometries. Units of class 1 appear to compare T-unit input from different sites on the body surface (Heiligenberg and Bastian 1980) whereas those of class 2 additionally appear to receive input from E- and I-units in the PLLL. Abbreviations: see MethodsThis study was supported by grants from the National Science Foundation and the National Institutes of Health.     

Ornithine decarboxylase prevents tumor necrosis factor alpha-induced apoptosis by decreasing intracellular reactive oxygen species

Ornithine decarboxylase (ODC) plays an essential role in various biological functions, including cell proliferation, differentiation and cell death. However, how it prevents the cell apoptotic mechanism is still unclear. Previous studies have demonstrated that decreasing the activity of ODC by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, causes the accumulation of intracellular reactive oxygen species (ROS) and cell arrest, thus inducing cell death. These findings might indicate how ODC exerts anti-oxidative and anti-apoptotic effects. In our study, tumor necrosis factor alpha (TNF-) induced apoptosis in HL-60 and Jurkat T cells. The kinetic studies revealed that the TNF- -induced apoptotic process included intracellular ROS generation (as early as 1 h after treatment), the activation of caspase 8 (3 h), the cleavage of Bid (3 h) and the disruption of mitochondrial membrane potential ( _m) (6 h). Furthermore, ROS scavengers, such as glutathione (GSH) and catalase, maintained _m and prevented apoptosis upon treatment. Putrescine and overexpression of ODC had similar effects as ROS scavengers in decreasing intracellular ROS and preventing the disruption of _m and apoptosis. Inhibition of ODC by DFMO in HL-60 cells only could increase ROS generation, but did not disrupt _m or induce apoptosis. However, DFMO enhanced the accumulation of ROS, disruption of _m and apoptosis when cells were treated with TNF- . ODC overexpression avoided the decline of Bcl-2, prevented cytochrome c release from mitochondria and inhibited the activation of caspase 8, 9 and 3. Overexpression of Bcl-2 maintained _m and prevented apoptosis, but could not reduce ROS until four hours after TNF- treatment. According to these data, we suggest that TNF- induces apoptosis mainly by a ROS-dependent, mitochondria-mediated pathway. Furthermore, ODC prevents TNF- -induced apoptosis by decreasing intracellular ROS to avoid Bcl-2 decline, maintain _m, prevent cytochrome c release and deactivate the caspase cascade pathway.     

Physiological comparison of d-cysteine desulfhydrase of Escherichia coli with 3-chloro-d-alanine dehydrochlorinase of Pseudomonas putida CR 1-1

d-Cysteine desulfhydrase of Escherichia coli W3110 trpED102/F trpED102 was physiologically characterized. It was found to be located in the cytosolic fraction, as 3-chloro-d-alanine dehydrochlorinase is. d-Cysteine desulfhydrase catalyzed not only the ,-elimination reaction of O-acetyl-d-serine to form pyruvate, acetic acid and ammonia, but also the -replacement reaction of O-acetyl-d-serine with sulfide to form d-cysteine. However, these reactions appeared not to proceed in vivo. No other activity of d-cysteine synthesis from O-acetyl-d-serine and sulfide was detected in a crude cell extract of E. coli which was immunotitrated with antibodies raised against the purified d-cysteine desulfhydrase. Although d-cysteine desulfhydrase catalyzes the degradation (,-elimination reaction) of 3-chloro-d-alanine, which is an effective antibacterial agent, E. coli W3110 trpED102/F trpED102 did not show resistance against 3-chloro-d-alanine. Therefore, d-cysteine desulfhydrase does not contribute to 3-chloro-d-alanine detoxification in vivo.     

SCM2, a tryptophan permease in Saccharomyces cerevisiae, is important for cell growth

SCM2, a novel gene encoding a yeast tryptophan permease, was cloned as a high-copy-number suppressor of cse2-1. The cse2-1 mutation causes cold sensitivity, temperature sensitivity and chromosome missegregation. However, only the cold-sensitive phenotype of cse2-1 cells is suppressed by SCM2 at high copy. SCM2 is located on the left arm of yeast chromosome XV, adjacent to SUP3 and encodes a 65 kDa protein that is highly homologous to known amino acid permeases. Four out of five disrupted scm2 alleles (scm21-4) cause slow growth, whereas one disrupted allele (scm25) is lethal. Cells with both the scm21 and trp1-101 mutations exhibit a synthetic cold-sensitive phenotype and grow much more slowly at the permissive temperature than cells with a single scm21 or trp1-101 mutation. A region of the predicted SCM2 protein is identical to the partial sequence recently reported for the yeast tryptophan permease TAP2, indicating that SCM2 and TAP2 probably encode the same protein.     

A gal region mutant that requires cAMP for growth on galactose in an adenyl cyclase negative (cya delta) background.

Summary Strains ofEscherichia coli K12 that contain a deletion of the adenyl cyclase gene (cya), required for the synthesis of cyclic adenosine-3; 5 monophosphate (cAMP), grow on galactose-containing minimal medium. A mutant was isolated that grows on this medium only if cAMP is added. The mutation (designatedgalP20) is linked to thegal operon region as determined by both generalized transduction with bacteriophage P1 and specialized transduction with bacteriophage . Studies withgalP20 cya strains as well asgal (deletions of thegal operon)cya strains indicate that synthesis of the physiologically important transport mechanism for galactose (galactose permease) requires either cAMP or a function missing from both thegal strains and thegalP20 strain.     

A novel eye morphology induced by a P element in somatic tissue of Drosophila melanogaster.

Summary We found a specific eye morphology designated as Square, which is induced when some Drosophila melanogaster strains harboring P elements are crossed with the 2–3 strain carrying a modified P element, P[ry ⁺, 2–3], which produces transposase in somatic tissue. This phenotype was dominant and also induced in the reciprocal crosses. Square was induced when the 2–3 strain was crossed with Q and M strains such as the sn^w (M) strain carrying three small P elements but not with P strains. Inheritance of Square was also tested and its phenotype was not transmitted to the next generation. These results suggest that Square is caused by the transposition of P elements in somatic cells.     

Bacteriophage phi 1 as a gene-cloning vector in Bacillus subtilis

Summary We attempted to use Bacillus subtilis phage 1 as a gene-cloning vector since the 1 genome was found to have few cleavage sites upon digestion with several kinds of restriction endonucleases. A 1 stock supplied by J. Ito (University of Arizona, Tucson, USA) consisted of two phages, 1E1 and 1E2, having one and two EcoRI-cleavage sites in their genomes respectively. From the latter isolate a deletion mutant 1E21 was induced to increase the size range of DNA segments to be cloned. It was demonstrated, by in vitro recombination experiments with phage 11 DNA, that 1E21 can be used for cloning EcoRI fragments of various sizes. We analyzed the DNAs of ten 1 clones isolated from independent transfectants and found that six of them carried 11 DNA fragments inserted at either of the two EcoRI-cleavage sites. Some of the hybrid phage DNAs were found to be cleaved with BamHI and HaeIII endonucleases at the 11 DNA portion, whereas the parental 1E21 DNA was insensitive to any of these enzymes. These hybrid phages would therefore be useful vectors for cloning foreign DNA fragments generated by cleavage with BamHI or HaeIII endonucleases.     

Effects of competition and N and P supply on carbon isotope discrimination and <Superscript>15</Superscript>N-natural abundance in four grassland species

The effect of interspecific competition and element additions (N and P) on four grassland species (Poa pratensis, Lolium perenne, Festuca valida, Taraxacum officinale) grown under field conditions was studied. Two grasses (L. perenne, F. valida) grown in monoculture (absence of competition) showed lower carbon isotope discrimination (¹³C) and enriched ¹⁵N values. Nitrogen addition (as urea) had inconsistent effects on species ¹³C while caused enrichment of ¹⁵N of P. pratensis and F. valida but strong depletion of ¹⁵N of T. officinale. Phosphorous had no significant effect on ¹³C but depleted ¹⁵N of all species.     

A suitable method for construction and cloning hybrid plasmids containing EcoRI-fragments of E. coli genome.

Summary A convenient procedure for the isolation of specificEcoRI-fragments ofE. coli genome and their amplification on Km-resistance plasmid vector CK 11 is described. The hybrid molecules were constructedin vitro usingEcoRI-digestion, followed by ligation. Then appropriatedE. coli strain was transformed with ligated DNA mixture and hybrid plasmids CK 11-arg ⁺, CK 11-his ⁺, CK 11-thr ⁺ and CK 11-leu ⁺ containing loci ofE. coli genome were selected by molecular cloning. The hybrid plasmids obtained consisted of oneEcoRI-fragment of initial plasmid CK 11 and one respective specific portion ofE. coli genome.