Catabolite repression in the D-serine deaminase system of Escherichia coli K-12

The induced synthesis of d-serine deaminase in Escherichia coli is subject to three catabolic effects: inhibition on inducer uptake, transient repression, and catabolite repression. Inhibition on d-serine uptake is not significant at the d-serine concentration normally used for induction. Transient repression and catabolite repression of d-serine deaminase synthesis are abolished by mutations in dsdCy, which appears to be an operator locus. The decline in the rate of constitutive synthesis observed in dsdCx mutants growing with glycerol as carbon source at temperatures above 37 C is due to catabolite repression. The low level of constitutivity at 37 C and the partial cis dominance of dsdCx mutants are not artifacts of catabolite repression. It is suggested that a product of one of the genes of the dsd operon may regulate the expression of the operon.     

Dominance studies with stable merodiploids in the D-serine deaminase system of Escherichia coli K-12

An episome, F32, which carries the genetic markers dsdA(+), the presumed structural gene for d-serine deaminase, dsdC(+), a regulatory locus governing the synthesis of d-serine deaminase, aroC(+), and purC(+) was obtained from strain AB311 of Escherichia coli K-12, and was used to construct appropriate merodiploids with dsdC markers. In all dsdC / dsdC(+) diploids examined, dsdC was found to be cis dominant, trans recessive, to dsdC(+). In two cases, however, the cis dominance was only partial. Moreover, complementation was observed between one of the dsdC markers which is fully cis dominant and one which is partially cis dominant. Because of the size of the dsdC region, the phenotypes of the mutants, and the partial trans dominance of dsdC(+) over some of the dsdC mutations, it is suggested that the dsdC region specifies a product, but that this product does not move with facility through the cytoplasm     

Transfer of a gene for sucrose utilization into Escherichia coli K12, and consequent failure of expression of genes for D-serine utilization.

As the first stage in investigating the genetic basis of natural variation in Escherichia coli, the gene(s) conferring the ability to use sucrose as a carbon and energy source (given the symbol sac+) was transferred from a wild strain to K12, which does not use sucrose. The sac+ region was transferred by two different methods. On both occasions it took a chromosomal location at minute 50.5 on the linkage map, between aroC and supN, in the region of the dsd genes, which confer the ability to use D-serine as a carbon and energy source. When the sac+ region was present in the K12 chromosome the bacteria were unable to use D-serine as a carbon and energy source. In F' sac+/dsd+ diploids, the dsd+ genes were similarly not expressed. Strain K12(sac+) bacteria were sensitive to inhibition by D-serine; they mutated to D-serine resistance with much greater frequency than did a dsd mutant of K12. Such bacteria also mutated frequently to use raffinose. Strain K12(sac+) bacteria did not utilize sucrose when they carried a mutation affecting the phosphotransferase system.     

Transient repression of the lac operon - the effect of a lac promoter deletion

Experiments have been done to show whether the lac promoter delection L1, which partly alleviates catabolite repression, also affects transient repression of lac. In stain L1/F'M15 all of the beta-galactosidase is synthesized from a chromosomal gene cis to L1, whereas 98% of the thiogalactosidase transacetylase is synthesized from an episomal gene cis to an intact i-p-o region. The addition of glucose to induced cultures of strain L1/F'M15 growing in glycerol medium caused extensive transient repression of transacetylase but almost no transient repression of beta-galactosidase. In control experiments with a diploid stain of genotype p(+)z(+)a(-)/F'p(+)z(-)a(+) the two enzymes suffered equal transient repression. Thus L1 substantially relieves transient repression.     

The Mutation Masculinizer (Man) Defines a Sex-Determining Gene with Maternal and Zygotic Functions in Musca Domestica L

In Musca domestica, the primary signal for sex determination is the dominant factor M, which is assumed to regulate a postulated female-determining gene F. Presence of M prevents expression of F so that male development ensues. In the absence of M, F can become active, which dictates the female pathway. The existence of F is inferred from F(D), a dominant factor that is epistatic to M. We describe a new mutation masculinizer, which has all the properties expected for a null or strongly hypomorphic allele of F: (1) it maps to the same chromosomal location as F(D), (2) homozygous man/man animals develop as males, (3) homozygous man/man clones generated in man/+ female larvae differentiate male structures, (4) man has a sex-determining maternal effect. About a third of the morphological males synthesize yolk proteins, which indicates that they are intersexual in internal structures. The maternal effect of man is complete in offspring that derive from homozygous man/man pole cells transplanted into female hosts. In this case, all man/+ progeny become fertile males that do not produce yolk proteins. A sex-determining maternal effect has previously been demonstrated for F(D). Like F, maternal man(+) is needed for zygotic man(+) to become active, providing further evidence that man is a loss-of-function allele of F.     

cis,cis-Muconate cyclase from Trichosporon cutaneum.

The inducible enzyme catalysing the conversion of cis,cis-muconate to (+)-muconolactone was purified 300-fold from the yeast Trichosporon cutaneum, grown on phenol. The enzyme has a sharp pH optimum at pH 6.6. It reacts also with several monohalogen derivatives and with one monomethyl derivative of cis,cis-muconate, but not with cis,trans- or trans,trans-muconate or 3-carboxy-cis,cis-muconate. In contrast with the corresponding enzymes in bacteria, the yeast enzyme does not require added divalent metal ions for activity and is not inhibited by EDTA. The purified enzyme can be resolved into two peaks by isoelectric focusing. The two forms have pI 4.58 (cis,cis-muconate cyclase I) and pI 4.74 (cis, cis-muconate cyclase II), respectively. Each of these is homogenous on polyacrylamide-gel electrophoresis in the absence or presence of sodium dodecyl sulphate. The two enzyme forms have the same molecular weight (50000) as determined by gel filtration and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. They have the same Km value (25 microM) for cis,cis-muconate. They differ with respect to their content of free thiol groups. cis, cis-Muconate cyclase I contains one thiol group, essential for activity, but relatively stable upon storage. cis, cis-Muconate cyclase II contains two thiol groups that are readily oxidized during storage with concomitant loss of activity.     

Exchange of genetic markers at extremely high temperatures in the archaeon Sulfolobus acidocaldarius.

When cells of two auxotrophic mutants of Sulfolobus acidocaldarius are mixed and incubated on solid medium, they form stable genetic recombinants which can be selected, enumerated, and characterized. Any of a variety of auxotrophic markers can recombine with each other, and the phenomenon has been observed at temperatures of up to 84 degrees C. The ability to exchange and recombine chromosomal markers appears to be an intrinsic property of S. acidocaldarius strains. It occurs between two cell lines derived from the same parent or from different parents and also between a recombinant and its parent. This is the first observation of chromosomal marker exchange in archaea from geothermal environments and provides the first functional evidence of generalized, homologous recombination at such high temperatures.     

D-serine deaminase is a stringent selective marker in genetic crosses.

The presence of the locus for D-serine deaminase (dsd) renders bacteria resistant to growth inhibition by D-serine and enables them to grow with D-serine as the sole nitrogen source. The two properties permit stringent selection in genetic crosses and make the D-serine deaminase gene an excellent marker, especially in the construction of strains for which the use of antibiotic resistance genes as selective markers is not allowed.     

Heteroduplex analysis of tra delta f'' plasmids and the mechanism of their formation.

Four tra delta FargG+ plasmids, derived from matings between Hfr AB312 and a recA recipient, have been shown to have deletions of at least 50% of the F genome, including the region in which the tra genes map. The mutant plasmids do contain the F genes required for plasmid maintenance. Correlations can be made between, on the one hand, the F genes present on the tradelta F' plasmids and the F genes transferred early by an Hfr donor, and, on the other hand, the F genes deleted from the tradelta F' plasmids and the F genes transferred late by an Hfr donor. A biased representation of proximally and distally transferred chromosomal markers among the tradelta F' elements was also demonstrated. Taken Taken together, the asymmetrical representation of Hfr genes and the cis dominance of the Tra phenotype of these mutants can best be explained by the hypothesis that the tradelta F' plasmids are formed by repliconation of the transferred exogenote in a recA recipient.     

Physical mapping of the Escherichia coli D-serine deaminase region: contiguity of the dsd structural and regulatory genes.

The genes dsdA, dsdO, and dsdC have been located on a 3.0-kilobase pair (kb) fragment of the Escherichia coli chromosome by a combination of techniques. The loci were first cloned onto lambda and various plasmid vectors. dsd hybrid plasmids were then digested with restriction enzymes, and the fragments were recloned to test for the presence of dsdC or dsdA. In one case, a 4.2-kb restriction fragment containing the dsdA operon was used to form a heteroduplex with a well-defined lambda dsd deoxyribonucleic acid. The results show that dsdA, dsdO, and at least 0.6 kb of dsdC are present on this piece of deoxyribonucleic acid. On the basis of the mapping analysis and the molecular weight of D-serine deaminase, 1.9 kb of the 4.2-kb fragment is accounted for by the three dsd loci. We conclude that dsdO and dsdC are contiguous. A detailed dsd restriction map is presented.     

Transvection and silencing of the Scr homeotic gene of Drosophila melanogaster

The Sex combs reduced (Scr) gene specifies the identities of the labial and first thoracic segments in Drosophila melanogaster. In imaginal cells, some Scr mutations allow cis-regulatory elements on one chromosome to stimulate expression of the promoter on the homolog, a phenomenon that was named transvection by Ed Lewis in 1954. Transvection at the Scr gene is blocked by rearrangements that disrupt pairing, but is zeste independent. Silencing of the Scr gene in the second and third thoracic segments, which requires the Polycomb group proteins, is disrupted by most chromosomal aberrations within the Scr gene. Some chromosomal aberrations completely derepress Scr even in the presence of normal levels of all Polycomb group proteins. On the basis of the pattern of chromosomal aberrations that disrupt Scr gene silencing, we propose a model in which two cis-regulatory elements interact to stabilize silencing of any promoter or cis-regulatory element physically between them. This model also explains the anomalous behavior of the Scx allele of the flanking homeotic gene, Antennapedia. This allele, which is associated with an insertion near the Antennapedia P1 promoter, inactivates the Antennapedia P1 and P2 promoters in cis and derepresses the Scr promoters both in cis and on the homologous chromosome.     

Effect of point mutations in the lac promoter on transient and severe catabolite repression of the lac operon of Escherichia coli

1. Experiments were devised to show whether the point mutations L8 and L29 in the lac promoter alleviate transient repression. 2. Several recombinants were picked from matings between a single F(-)p(+) strain and Hfr strains carrying mutations L8 and L29. All of the 19 p(-) recombinants tested proved to suffer no transient repression, whereas all of the eight p(+) recombinants tested suffered prolonged transient repression. 3. A diploid strain was constructed in which more than 90% of the thiogalactoside transacetylase is synthesized from the episome with a wild-type lac promoter, whereas 100% of the beta-galactosidase is synthesized from the chromosome with a promoter carrying mutation L8. In this diploid the synthesis of thiogalactoside transacetylase suffered transient repression but the synthesis of beta-galactosidase did not. 4. Exactly similar results were obtained with a diploid strain in which the chromosomal promoter carried mutation L29. 5. The same diploid strains were used in experiments to show whether mutations L8 and L29 alleviate the severe catabolite repression caused by growth in glucose plus gluconate. In both strains glucose+gluconate repressed the synthesis of beta-galactosidase much less than the synthesis of thiogalactoside transacetylase. 6. These and previously reported results can be explained by assuming (a) that both mutations L8 and L29 render the lac promoter partially, but not completely, insensitive to catabolite repression, and (b) that transient repression is an exceptionally severe form of catabolite repression.     

ColB2-K77, a Fertility-Repressed F-Like Sex Factor

The colicinogenic B factor, transferred from Escherichia coli strain K77 (and termed ColB2-K77 or ColB2) to an E. coli K12 F(-) strain, is capable of promoting its own transfer to other K12 F(-) strains at a low rate (from LFC cultures) which can be increased under special conditions (HFC cultures). LFC cultures of K12 (ColB2)(+) F(-) strains show a low level of adsorption of F-specific phage particles which also increases under HFC conditions. The ColB2 factor is thus inferred to be an F-like sex factor which is repressed in its fertility. This repression is concluded to be due to a cytoplasmic repressor since, when ColB2 is present in cells containing an F factor (either autonomous or integrated), F fertility is also repressed as shown by the inability of such (ColB2)(+)F(+) [or (ColB2)(+)Hfr] strains to plaque F-specific phages, and by a reduction in the level of chromosomal transfer from such strains, compared to the corresponding F(+) (or Hfr) control strains. Mutants of the ColB2 factor in which fertility is no longer repressed (fertility derepressed or Fdr mutants) have been isolated. The ColB2Fdr mutant strains do not appear to be able to mobilize chromosomal transfer, although they have acquired F-specific phage sensitivity demonstrable by plaque formation and they transfer their colicin factor at high frequency and are well piliated. The Fdr mutation is presumed to result in the inability to synthesize the cytoplasmic fertility repressor since the ColB2Fdr factor does not repress the fertility of an F factor when present in the same host strain. A fertility-repressed drug resistance factor of the R(f) type is not stable in the presence of a ColB2 factor in the same cell and is eliminated in about 10% of the cells per generation. In contrast, another factor characteristic of the R(i) type is fully compatible with ColB2. Under conditions artificially stabilizing (ColB2Fdr)(+) (Rf)(+) strains, the enhanced fertility of ColB2Fdr is not repressed by the presence of the R factor, nor does the presence of R(f) in the intermediate strain of an HFC (for ColB2) system inhibit the normal increase in ColB2 transmissibility. It is concluded that the repressors of R(f) and ColB2, although both active on F fertility, are different; this may indicate that at least two independently repressible cistrons are involved in the expression of fertility characteristics.     

Stereoselective metabolism of the (+)-(S,S)- and (-)-(R,R)-enantiomers of trans-3,4-dihydroxy-3,4-dihydrobenzo[c]-phenanthrene by rat and mouse liver microsomes and by a purified and reconstituted cytochrome P-450 system

Metabolism of (+)-, (-)-, and (+/-)-trans-3,4-dihydroxy-3, 4-dihydrobenzo[c]phenanthrenes by liver microsomes from rats and mice and by a purified monooxygenase system reconstituted with cytochrome P-450c has been examined. Bay-region 3,4-diol 1,2-epoxides are minor metabolites of both enantiomers of the 3,4-dihydrodiol with liver microsomes from 3-methylcholanthrene-treated rats or with the reconstituted system (less than 10% of total metabolites). Microsomes from control and phenobarbital-treated rats and from control mice form higher percentages of these diol epoxides (13-36% of total metabolites). Microsomes from 3-methylcholanthrene-treated rats and cytochrome P-450c in the reconstituted system form exclusively the diol expoxide-1 diastereomer, in which the benzylic hydroxyl group and oxirane oxygen are cis to each other, from the (+)-(3S,4S)-dihydrodiol. The same enzymes selectively form the diol expoxide-2 diastereomer, with its oxirane oxygen and benzylic hydroxyl groups trans to each other, from the (-)-(3R,4R)-dihydrodiol (77% of the total diol epoxides). Liver microsomes from control rats show similar stereoselectivity whereas liver microsomes from phenobarbital-treated rats and from control mice are less stereoselective. Three bis-dihydrodiols and three phenolic dihydrodiols are also formed from the enantiomeric 3,4-dihydrodiols of benzo[c]phenanthrene. A single diastereomer of one of these bis-dihydrodiols with the newly introduced dihydrodiol group at the 7,8-position accounts for 79-88% of the total metabolites of the (-)-(3R,4R)-dihydrodiol formed by liver microsomes from 3-methylcholanthrene-treated rats or by the reconstituted system containing epoxide hydrolase. In contrast, the (+)-(3S,4S)-dihydrodiol is metabolized to two diastereomers of this bis-dihydrodiol, a third bis-dihydrodiol, and two phenolic dihydrodiols.     

Changes in chromosomal polymorphism and global warming: The case of Drosophila subobscura from Apatin (Serbia)

In this study, chromosomal inversion polymorphism data for a natural population of Drosophila subobscura from a swampy region near the town of Apatin (Serbia) were compared with data for the same population collected approximately 15 years earlier. The pattern of chromosomal inversion polymorphism changed over time. There were significant increases in the frequency of characteristic southern latitude ("warm" adapted) chromosomal arrangements and significant decreases in the frequency of characteristic northern latitude ("cold" adapted) chromosomal arrangements in the O and U chromosomes. The chromosomal arrangements O(3+4) and O(3+4) (+) (22) (derived from the O(3+4) arrangement) showed significant increases in 2008 and 2009 with regard to the 1994 sample. There was also a significant increase (～50%) in the U(1) (+) (2) arrangement, while U(1+8) (+) (2) (a typical southern arrangement) was detected for the first time. Since the Apatin swampy population of D. subobscura has existed for a long time in a stable habitat with high humidity that has not been changed by man our results indicate that natural selection has produced chromosomal changes in response to the increase in temperature that has occurred in the Balkan Peninsula of central southeastern European.     

A Comparative Study of Two F-like Colicin Factors, ColV2 and ColV3, in Escherichia coli K-12

Of three colicin factors, each determining the synthesis of a colicin V in three wild-type Escherichia coli strains studied, two were shown to have sex-factor activities. In E. coli K-12, these activities resembled those of the F sex factor (including rapid and efficient self-transmission in exponentially growing cultures, adsorption of "male-specific" ribonucleic acid phage, production of "female phenocopies," elimination by acridine orange, and chromosomal transfer dependent upon recombination with host bacterium) and differed in this way from those of the colicin I sex factor (ColI). The two V factors, ColV2 and ColV3, differed in their efficiency of plating male-specific phage and in the pattern of transfer of chromosomal markers. Furthermore, although neither factor could stably coexist with F within the same cell, they showed markedly different exclusion effects. In general, ColV2 excluded F and ColV3 was excluded by F, irrespective of which sex factor was preestablished in the cell. An exception to this was the ability of ColV2 to stabilize in any one of a series of Hfr strains, giving rise to strains which in the majority of cases showed normal Hfr and colicinogenic properties.     

Cis proline mutants of ribonuclease A. I. Thermal stability.

A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol. 185, 60-89). The expressed protein, which contains an additional N-terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A. The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification. Site-directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made. In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing. Thermal unfolding experiments on four single mutants give delta Tm approximately equal to 10 degrees C and delta delta G0 (apparent) = 2-3 kcal/mol. The reason is that either the substituted amino acid goes in cis, and cis<==>trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild-type conformation, that allows the substituted amino acid to form a trans peptide bond.     

Episome-carried surface antigen K88 of Escherichia coli. I. Transmission of the determinant of the K88 antigen and influence on the transfer of chromosomal markers

?rskov, Ida (Statens Seruminstitut, Copenhagen, Denmark), and Frits ?rskov. Episome-carried surface antigen K88 of Escherichia coli. I. Transmission of the determinant of the K88 antigen and influence on the transfer of chromosomal markers. J. Bacteriol. 91:69-75. 1966.-The transmission of the determinant of the Escherichia coli K88 antigen in mixed cultures of E. coli strains is described. The K88 factor could not be transferred by filtrates, nor could responsible phages or colicines be detected. Acriflavine was shown to "cure" the bacteria for the K88 antigen. Generally, the strains having acquired the K88 antigen also acquired the ability to transfer chromosomal markers, but this ability was in some cases retained by segregants which had lost the K88 antigen. Introduction into an F(+) strain caused reduction of the recombination frequency and disappearance of the f(+) antigen. Not all wild-type strains with the K88 antigen are genetic donors of this antigen, at least not to a discernible degree. It was concluded that the K88 antigen determinant is carried by an episome.     

Excess heterozygosity contributes to genetic map expansion in pea recombinant inbred populations

Several plant genetic maps presented in the literature are longer than expected from cytogenetic data. Here we compare F(2) and RI maps derived from a cross between the same two parental lines and show that excess heterozygosity contributes to map inflation. These maps have been constructed using a common set of dominant markers. Although not generally regarded as informative for F(2) mapping, these allowed rapid map construction, and the resulting data analysis has provided information not otherwise obvious when examining a population from only one generation. Segregation distortion, a common feature of most populations and marker systems, found in the F(2) but not the RI, has identified excess heterozygosity. A few markers with a deficiency of heterozygotes were found to map to linkage group V (chromosome 3), which is known to form rod bivalents in this cross. Although the final map length was longer for the F(2) population, the mapped order of markers was generally the same in the F(2) and RI maps. The data presented in this analysis reconcile much of the inconsistency between map length estimates from chiasma counts and genetic data.     

CONTACT-INHIBITED REVERTANT CELL LINES ISOLATED FROM SV40-TRANSFORMED CELLS : II. Ultrastructural Study

The ultrastructural appearances of normal 3T3, SV40-transformed 3T3 (SV-3T3), and F1A revertant cell lines are compared. Both confluent and subconfluent cultures are described after in situ embedding of the cells for electron microscopy. There is striking nuclear pleomorphism in F1A revertant cells, with many cells having large nuclei compared to the less variable nuclear morphology of both normal 3T3 and SV-3T3 cells. Under the culture conditions used, deep infoldings of the nuclear envelope are prominent in growing cells, e.g., subconfluent normal 3T3 and confluent SV-3T3 cells. Such infoldings are infrequently seen in cultures which display contact inhibition of growth, e.g., normal 3T3 or F1A revertant cells grown just to confluence. In confluent cultures, the cytoplasmic organelles in revertant cells closely resemble those of normal 3T3 cells. In both normal and revertant cells in confluent culture, the peripheral cytoplasm (ectoplasm) has many 70 A filaments (alpha filaments), which are frequently aggregated into bundles. Alpha filaments are also abundant in the ectoplasm near regions of cell-to-cell apposition and in the motile cell processes (filopodia). The abundance and state of aggregation of alpha filaments correlates with contact inhibition of movement and growth in these cell lines since fewer bundles of alpha filaments are seen in growing cells than in contact-inhibited cells. This observation suggests that these filaments may be an important secondary component in the regulation of contact inhibition of movement and, possibly, of growth in normal and revertant cells.