Arsenate-resistant alkaline phosphatase-constitutive mutants of Escherichia coli.

Summary When arsenate-resistant mutants are selected approximately 50 per cent of them are also consitutive for the synthesis of alkaline phosphatase and the Pi-binding protein. Some of these mutants are linked to ilv (phoS ^- or phoT ^-), others are linked to proC (phoR ^-). One of the mutant strains linked to ilv lost the Pi-binding protein (the phoS gene product). Resistance to arsenate, constitutivty for alkaline phosphatase synthesis and loss of the Pi-binding protein occurred pleiotropically by the same phoS ^- mutation.     

Production of extracellular alkaline phosphatase by Escherichia coli K-12 periplasmic-leaky mutants carrying phoA + Plasmids

Summary Col E1 hybrid plasmids carrying the phoA ⁺ structural gene of alkaline phosphatase, a periplasmic enzyme of Escherichia coli K-12, were identified from the Clarke and Carbon genomic bank. Wild-type (lky ⁺) phoA ⁺ plasmid-bearing strains synthesized 14 times more intracellular enzyme than the haploid lky ⁺ strain. Phosphate-induced repression was maintained in transformed strains. PhoA ⁺ plasmids carrying the phoB and phoR regulatory genes were introduced into a periplasmic-leaky (lky) recipient strain able to release alkaline phosphatase into the extracellular medium. Transformed lky mutants excreted up to 90% of total enzyme activity which corresponded to 3.5 times the amount of intracellular alkaline phosphatase made by the haploid lky ⁺ strain. The protein composition analysis of periplasmic and extracellular fractions showed that: (i) wild-type phoA ⁺ hybrid plasmidbearing clones did not excrete alkaline phosphatase but had a modified periplasmic content; (ii) alkaline phosphatase was the major excreted protein by transformed lky mutants. The use of periplasmic-leaky phoA ⁺ hybrid plasmid-bearing mutants for an easier production and purification of alkaline phosphatase is discussed.     

Apparent non-involvement of transfer RNA nucleotidyltransferase in the biosynthesis of Escherichia coli suppressor transfer RNAs

Transfer RNA nucleotidyltransferase has previously been shown to be required for the normal growth of Escherichia coli and for the biosynthesis of some bacteriophage T4 tRNAs. In order to obtain information about the involvement of this enzyme in E. coli tRNA biosynthesis we have measured the level of activity of suppressors 1 to 6 in strains carrying either a cca⁺ or cca allele. We found that cca strains, deficient in tRNA nucleotidyltransferase, contained the same amount of suppressor activities as the wild-type cca⁺ strains as determined by suppression of nonsense mutations in both E. coli alkaline phosphatase and in genes of bacteriophage T4. The results suggest that tRNA nucleotidyltransferase is not required for the biosynthesis of tRNAs specified by suppressors 1 to 6.     

A new locus in the phosphate specific transport (PST) region of Escherichia coli

Summary PhoS64 is a mutation in the Phosphate Specific Transport (PST) region on the E. coli chromosome which lacks the periplasmic phosphate binding protein. In contrast to other phoS mutations (which have the same phenotype) it complements the mutations in phoT and pstB. A detailed genetic map of the PST region constructed by three point transductional crosses has revealed that phoS64 is located distally from other phoS mutations. The genetic order obtained was phoS64-phoU35-pstB401-phoT-phoS-ilvC. The data indicate that phoS64 belongs to a different complementation unit in the PST region not known hitherto. We propose to name it phoV.Abbreviations AP alkaline phosphatase - EU enzyme units - Pi inorganic orthophosphate - pNPP paranitrophenyl phosphate - Km kanamycine - Tc^r tetracycline-resistant     

Growth instability of the alkaline phosphatase repressor

Inhibition of DNA synthesis does not prevent thermal derepression of alkaline phosphatase in a phoS^tss mutant of Escherichia coli.     

Revertants from RNase III negative strains of Escherichia coli

Summary E. coli strains carrying the rnc-105 allele do not show any level of RNase III in extracts, grow slower than rnc ⁺ strains at temperatures up to 45°C and fail to grow at 45°C. Revertants which can grow at 45°C were isolated. The vast majority of them still do not grow as fast as rnc ⁺ strains and did not regain RNase III activity. The mutation(s) which caused them are suppressor mutations (physiological suppressors) which do not map in the immediate vicinity of the rnc gene. A few of the revertants regain normal growth, and contain normal levels of RNase III. They do not harbor the rnc-105 allele and therefore are considered to be true revertants. By using purines other than adenine it was possible to isolate rnc ⁺ pur ^- revertants from an rnc ^- pur ^- strain with relative ease. They behaved exactly like the true rnc ⁺ revertants isolated from rnc ^- strains at 45°C.A merodiploid strain which contains the rnc ⁺ gene on an episome behaves exactly like an rnc ⁺ strain with respect to growth and RNA metabolism, eventhough its specific RNase III activity is about 60% of that of an rnc ⁺ strain; thus the level of RNase III is not limiting in the cell.The rnc ^- strains show a characteristic pattern of transitory molecules, related to rRNA, 30S, 25S, p23 and 18S, which are not observed in rnc ⁺ strains. This pattern is unchanged in rnc ^- strains and in the revertants which are still lacking RNase III, regardless of the temperature in which RNA synthesis was examined (30° to 45°C). On the other hand, in the rnc ⁺ strains as well as in the true revertants and the rnc ⁺/rnc ^- merodiploid, the normal pattern of p16 and p23 is observed at all temperatures. These findings suggest that all the effects observed in RNase III^- strains are due to pleiotropic effects of the rnc-105 allele, and that the enzyme RNase III is not essential for the viability of the E. coli cell.     

Genetic and endocrine control of renin activity in the submaxillary gland of the mouse

Basal activity of submaxillary gland (SMG) renin is high in female mice that carry the Rnr ^s allele and is induced to higher levels by treatment with dihydrotestosterone (DHT). To determine whether the difference in basal activity between high (Rnr ^s/Rnr^s) and low (Rnr ^b/Rnr^b) strains is due to enhanced sensitivity of Rnr ^s/Rnr^s strains to endogenous androgen, we first studied the effect of several types of endocrine ablation on SMG renin in young female mice, and second, we removed normal androgen receptor protein by introducing the X-linked Tfm gene. Adrenalectomy with or without castration had no effect on basal SMG renin; hypophysectomy decreased basal renin activity 400-fold but did not abolish responsiveness to DHT. Loss of androgen receptor did not affect basal renin activity but did prevent enhancement by DHT. Basal and induced renin activities in L.AKR(Alll)/Cy, a congenic strain homozygous for Rnr ^s introduced from AKR/J into the background of C57L/J, an Rnr ^b/Rnr^b type strain, are intermediate between levels observed in the original strains. We conclude that (1) the basal level of SMG renin is regulated directly or indirectly by some pituitary hormone(s) but not by androgen, (2) androgen induction of renin activity requires a normal androgen receptor, and (3) major gene(s) that regulate basal as well as induced SMG renin are in a circumscribed region of chromosome 1.This work has been aided by Grants GM26414 and AM03892 from the National Institutes of Health, a grant from the Texas affiliate of the American Heart Association, and by research contract NO1-CP33255 from the Division of Cancer Cause and Prevention, the National Cancer Institute. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Overproduction and excretion ofβ-lactamase and alkaline phosphatase byEscherichia coli olp mutants

Summary We have isolated spontaneousolp mutants ofEscherichia coli K-12 overproducing the periplasmic enzymes -lactamase (Bla) and alkaline phosphatase (PhoA). Enzyme overproduction was maintained inolp strains transformed with plasmids carryingbla ⁺ andphoA ⁺ structural genes, and synthesizing high levels of Bla and PhoA. Transformedolp strains excreted up to 40% of these enzymes into the growth medium. The introduction of atolA excretory mutation intoolp strains led to an increase of enzyme overproduction and a release of 85% of Bla and PhoA enzyme activities into the culture medium.     

New complexities at the Qa-1 locus

Mouse strain and tissue distribution analyses indicate that the new antiserum A anti-A-Tla ^b recognizes the cell-surface product governed by the previously serologically undetectable Qa-I ^b allele. This cell-surface product has therefore been called Qa-1.2. Three levels of anti-Qa-1.2 cytotoxicity in the presence of complement have been observed: high, intermediate, and zero lysis. In general, high levels of lysis correlate with the presence of the Qa-1 b allele, while zero levels of lysis correlate with the presence of the Qa-1 ^aallele. The A.CA strain reacts with both anti-Qa-1.1 and anti-Qa-1.2 and may possess a third allele, Qa-1 ^d. Several strains including B6-H-2 ^k react in an intermediate fashion. Recombinant strain analyses indicate that this intermediate reaction may be due to modifying genes within the H-2D region.     

The isolation and characterization of a mutant allele at a new X-linked locus,mex, affecting NADP+-dependent enzymes inDrosophila melanogaster

The isolation and characterization of mutant alleles in a regulatory gene affecting NADP⁺-dependent enzymes are described. The locus,mex, is at position 26.5 ± 0.74 on the X chromosome ofDrosophila melanogaster. The newly isolated mutant allele,mex ¹, is recessive to either themex allele found in Oregon-R wild-type individuals or that found in thecm v parental stock in which the new mutants were induced. Themex ¹ mutant allele is associated with statistically significant decreases in malic enzyme (ME) specific activity and ME specific immunologically cross-reacting material (ME-CRM) in newly emerged adult males. During this same developmental stage in males, the NADP⁺-dependent isocitrate dehydrogenase specific activity increases to statistically significant levels. Females of themex ¹ mutant strain show statistically significant elevated levels of the pentose phosphate shunt enzymes, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Isoelectric focusing and thermolability comparisons of the active ME from mutant and control organisms indicate that the enzyme is the same. Developmental profiles ofmex ¹ and control strains indicate that this mutant allele differentially modulates the levels of ME enzymatic activity and ME-CRM during development. This work was supported by an Operating Grant from the Natural Sciences and Engineering Research Council of Canada to M.M.B.     

Genetics of alkaline phosphatase of the small intestine of the house mouse (Mus musculus)

Four inbred strains of mice exhibited either slow (PL/J), intermediate (DBA/2J, LP/J), or fast (SWR/J) rates of migration of duodenal alkaline phosphatase on cellulose acetate electrophoresis. Hybrids of these strains also had intermediate rates of migration regardless of the combination of strains used as parents. Strain differences were present in all regions of the small but not the large intestine. Crosses of the PL/J strain to hybrids between this strain and the other three strains gave a 1:1 segregation of the slow and intermediate patterns. The symbol Akp-3 is proposed for the locus responsible for the slower migration of the enzyme in this strain. Data from the LP/J × PL/J hybrid crossed with the PL/J strain showed linkage with two loci on chromosome 1 as follows: centromere—Idh-1–13.8±3.1 cM—Akp-3–8.9±2.6 cM—Pep-3. The available data do not reveal the genetic basis for the faster migration rate of the enzyme from the SWR/J strain, but a different response to neuraminidase and apparent nonlinkage to the Pep-3 locus suggest that a locus other than Akp-3 is responsible.This work was supported by a grant from the University Research Committee, Indiana State University.     

The effect of the rpoSam allele on gene expression and stress resistance in Escherichia coli

The RNA polymerase associated with RpoS transcribes many genes related to stationary phase and stress survival in Escherichia coli. The DNA sequence of rpoS exhibits a high degree of polymorphism. A C to T transition at position 99 of the rpoS ORF, which results in a premature amber stop codon often found in E. coli strains. The rpoSam mutant expresses a truncated and partially functional RpoS protein. Here, we present new evidence regarding rpoS polymorphism in common laboratory E. coli strains. One out of the six tested strains carries the rpoSam allele, but expressed a full-length RpoS protein owing to the presence of an amber supressor mutation. The rpoSam allele was transferred to a non-suppressor background and tested for RpoS level, stress resistance and for the expression of RpoS and sigma70-dependent genes. Overall, the rpoSam strain displayed an intermediate phenotype regarding stress resistance and the expression of σ^S-dependent genes when compared to the wild-type rpoS ⁺ strain and to the rpoS null mutant. Surprisingly, overexpression of rpoSam had a differential effect on the expression of the σ⁷⁰-dependent genes phoA and lacZ that, respectively, encode the enzymes alkaline phosphatase and β-galactosidase. The former was enhanced while the latter was inhibited by high levels of RpoSam.     

The effect of metal ions on the alkaline phosphatase of Rhizobium leguminosarum

The alkaline phosphatase (EC 3.1.3.1.) from Rhizobium leguminosarum WU235 has been purified. The enzyme is a non-specific phosphomonoesterase, has a molecular weight of 78,500 and a sub-unit molecular weight of 39,400. Magnesium and zinc ions are implicated in the structure of the enzyme; atomic absorption analysis gave 1.9 g-atoms Mg²⁺ and 1.9–5.1 g-atoms Zn²⁺ per mole of enzyme. In addition high concentrations of Mg²⁺ markedly stimulate the enzyme. The phosphatase is inhibited by Li⁺ and Na⁺ and stimulated by K⁺, Rb⁺ and Cs⁺, which suggests that the enzyme is K⁺ activated.     

EFFECT OF UNILATERAL VISUAL DEPRIVATION AND VISUAL STIMULATION ON THE ACTIVITIES OF ALKALINE PHOSPHATASE, ACID PHOSPHATASE, Na+−K+ ACTIVATED Mg2+ CATALYSED ADENOSINE TRIPHOSPHATASE AND ON THE CONTENT OF SODIUM AND POTASSIUM IONS OF THE OPTIC LOBE OF ADULT PIGEON

—A study was made of the effects of unilateral visual deprivation and stimulation upon the activities of alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2), Na⁺-K⁺ activated Mg²⁺ catalysed ATPase (EC 3.6.1.4) and upon the Na⁺ and K⁺ contents of the optic lobe of adult pigeon (Columba livia). Visual deprivation was achieved by eyelid suturing or by enucleation and maintained for 1–9 weeks. Unilateral visual stimulation was maintained for 75 min following 72 h of darkness. A statistically significant increase in the activity of alkaline phosphatase activity was observed in the optic lobe after unilateral visual deprivation whereas unilateral visual stimulation resulted in the opposite effect. Acid phosphatase activity was found to be unchanged under all experimental conditions. Na⁺-K⁺ ATPase activity was found to increase significantly following unilateral visual stimulation and following eyelid suturing in the corresponding optic lobes; unilateral enucleation resulted in a decrease in the Na⁺-K⁺ ATPase activity. An increase in the enzyme activity was found to be associated with an increase in the level of Na⁺-ion and a decrease in the level of K⁺-ion, and vice versa.     

Arginine regulon control in a Salmonella typhimurium--Escherichia coli hybrid merodiploid.

Summary The regulation of synthesis of arg enzymes was studied in a hybrid merodiploid in which an episome of Escherichia coli carrying the argR ⁺ allele was transferred to a Salmonella typhimurium argR strain. The arg enzyme levels of the hybrid merodiploid were compared to that found in argR and argR ⁺ haploids of S. typhimurium. The results showed that repression of synthesis of arg enzymes was effected through the introduction of the E. coli argR ⁺ allele but significant quantitative differences of arg enzyme levels in the argR ⁺ haploid and the hybrid merodiploid were observed.     

Analysis of merodiploids of the cysB region in Salmonella typhimurium.

Summary We have studied the regulation of two cysteine biosynthetic enzymes in S. typhimurium merodiploid strains which are heterozygous at the cysB regulatory locus. This gene codes for an element of positive control which is necessary for the expression of the enzymes of the biosynthetic pathway. Under conditions of sulfur deprivation levels of sulfite reductase (coded for by cysI, cysJ and cysG) and of O-acetylserine sulfhydrylase (coded for by cysK) are derepressed in cysB ⁺ haploid strains, but not in cysB ^- haploid strains. Growth on a rich sulfur source such as l-cystine results in low levels of both enzyme activities in cysB ⁺ and cysB ^- haploid strains but not in cysB ^c haploid strains, where enzyme expression is constitutive, i.e. substantially greater than in a cysB ⁺ strain grown on l-cystine, regardless of the nutrients used for growth.We find that cysB ^-/F cysB ⁺ merodiploid strains can be derepressed for sulfite reductase and O-acetylserine sulfhydrylase by growth on a poor sulfur source, and therefore cysB ⁺ is dominant to cysB ^-. Enzyme levels are also derepressed in l-cystine-grown cysB ^c/F cysB ⁺ strains indicating that cysB ^c is dominant to cysB ⁺. The cysB484 allele is known to be cysB ^- in regard to the regulation of sulfite reductase activity, but cysB ^c with respect to O-acetylserine sulfhydrylase. In a cysB484/F cysB ⁺ strain the cysB ^- character of cysB484 is recessive to cysB ⁺, while cysB ^c is dominant to cysB ⁺.Merodiploids of the type cysB ^-/F cysB ⁺, bearing chromosomal point mutations are derepressed by sulfur deprivation to levels which are either less than, equal to, or greater than those of wild type. These results can be explained by assuming a multimeric structure for the cysB protein and the formation in merodiploids of cysB ^-/cysB ⁺ hybrid molecules with altered capacities for gene activation. The dominance of cysB ^c over cysB ⁺ indicates that in contrast to the araC regulatory protein, which acts as both a gene activator and repressor, the cysB protein serves only as an element of positive control.     

Cloning of phoM, a gene involved in regulation of the synthesis of phosphate limitation inducible proteins in Escherichia coli K12

Summary Like the synthesis of alkaline phosphatase, the synthesis of outer membrane PhoE protein is shown to be dependent on the phoM gene product in phoR mutants of E. coli K12. This phoM gene has been cloned into the multicopy vector pACYC184 using selection for alkaline phosphatase constitutive synthesis in a phoR background. The gene was localized on the hybrid plasmids by analysis of deletion plasmids constructed in vitro and of mutant plasmids generated by insertions.Interestingly, two of the selected hybrid plasmids contained the entire phoA-phoB-phoR region of the chromosome, as a multiple copy state of these genes results in the constitutive synthesis of alkaline phosphatase. The presence of multiple copies of the phoM gene hardly influences the level of expression of alkaline phosphatase and PhoE protein in a pho ⁺ strain, but significantly increases the levels of these proteins in aphoR mutant strain.     

Genetic control of glucokinase activity in mice

Inbred strains of mice were surveyed for liver glucokinase activity. Mice of all strains studied could be distributed into three groups with high, intermediate, and low levels of enzyme activity. Genetic analysis using crosses and backcrosses with prototype high (C3H/HeJ) and low (RF/J) strains revealed that glucokinase activity was controlled by a single gene. The name glucokinase and gene symbol Gk are suggested for this gene. The Gk ^a allele designates the strain with high glucokinase activity, while Gk ^b represents the allele in the strain with the low enzyme activity. The interaction of fasting and diabetes on the activity of glucokinase in these two strains is described.Supported in part by United States Public Health Service Research Grant CA 05873 from the National Cancer Institute. The Jackson Laboratory is fully accredited by the American Association for the Accreditation of Laboratory Animal Care.     

A phosphatase of undefined function is common to the photoreceptive microvilli of several arthropod species

Summary It has previously been demonstrated, using an ultracytochemical technique, that the photoreceptive microvilli of crab retinae contain a magnesium-dependent phosphatase that hydrolyses the artificial substrate 4-nitrophenylphosphate. Whilst many phosphatases hydrolyse 4-nitrophenylphosphate, the properties of the microvillar enzyme indicated that it is not a conventional acid or alkaline phosphatase. Using the same technique, it is now shown that a similar activity resides in the rhabdomeric microvilli of both the lateral compound eye and the ventral photoreceptors of Limulus polyphemus as well as in the compound eyes of the freshwater crayfish Cherax destructor and the fly Lucilia cuprina. Control cytochemical procedures performed on crayfish and fly showed that in these species too the activity is magnesium-dependent and is not due to a Na⁺/K⁺ ATPase.     

Interactions between viomycin resistance and streptomycin resistance on ribosomes of Mycobacterium smegmatis.

We have investigated the mechanism of the expression of resistance to high levels of viomycin and coresistance to streptomycin in a mutant strain of Mycobacterium smegmatis ATCC 14468 (AC-13) which was obtained by serial transfers of parental cells to media containing increasing concentrations of viomycin. It was shown previously that resistance to viomycin by strain AC-13 was due to an alteration in the 50 S ribosomal subunit (20). However, genetic analysis has shown that mutation in 50 S subunits alone gave only low level resistance to viomycin. When a streptomycin resistant mutation (caused by an alteration in the 30 S subunit) was introduced into the low level viomycin resistant recombinant strains, most of them were highly resistant to viomycin. Some recombinants were resistant to intermediate levels of viomycin, and the remainder were not affected by the introduction of the str^r allele. Studies with in vitro cell-free systems have shown that streptomycin resistant 30 S ribosomal subunits obtained from a high level viomycin resistant recombinant were able to modify the levels of resistance to viomycin expressed by the 50 S ribosomal subunit. These findings provide additional evidence concerning the functional relationship between 30 S and 50 S ribosomal components in ribosomes.