Mutants of Escherichia coli defective in membrane phospholipid synthesis. Phenotypic suppression of sn-glycerol-3-phosphate acyltransferase Km mutants by loss of feedback inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Revertants of Escherichia coli mutants defective in the first enzyme of membrane phospholipid synthesis, sn-glycerol-3-phosphate (glycerol-P) acyltransferase, were investigated. These glycerol-P acyltransferase mutants, selected as glycerol-P auxotrophs, contained membranous glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than the parental activity. The glycerol-P acyltransferase activity was also more thermolabile in vitro than the parental activity. Most revertants no longer requiring glycerol-P for growth regained glycerol-P acyltransferase activity of normal thermolability and apparent Km for glycerol-P. However, two novel revertants were isolated which retained an abnormal glycerol-P acyltransferase activity. The glycerol-P dehydrogenase activities of these novel revertants were about 20-fold less sensitive to feedback inhibition by glycerol-P. The feedback-resistant glycerol-P dehydrogenase co-transduced with gpsA, the structural gene for the glycerol-P dehydrogenase. Further transduction experiments demonstrated that the feedback resistant glycerol-P dehydrogenase phenotypically suppressed the glycerol-P acyltransferase Km lesion. The existence of the class of glycerol-P auxotrophs which owe their phenotype to the glycerol-P acyltransferase Km lesion therefore depends on the feedback regulation of glycerol-P synthesis in E. coli.     

Study of an H-2Kd mutant strain: C.B6-H-2dm4

A new H-2 mutant involving the H-2d haplotype is described--C.B6-H-2dm4 (dm4). This mutant strain carries a gain and loss mutation which maps to the Kd gene of the H-2 complex. Serological testing comparing the mutant and the parental BALB/cKh strain failed to detect any difference between the two strains and no antibodies could be produced, although a reciprocal mixed lymphocyte reaction was observed between mutant and parent.     

Mapping of new recessive cataract gene (itIr2) in the mouse

A new strain of mice with cataracts was developed in BALB/cHeA and STS/A recombinant inbred strain, CXS4 (D). In this study the mapping of spontaneous autosomal recessive cataract mutation is described. This mutation was characterized by ruptures of the lens nucleus, vitreous chamber through the posterior capsule, and the vacuolization of the lens. For the linkage analysis, we produced two kinds of backcross progenies, (BALB/cHeA × D)F₁ and (STS/A × D)F₁ females crossed to D male mice. The gene (lr2, lens rupture2) was mapped to the central part of Chromosome(Chr) 14, 0.7 ± 0.7cM from the micosatellite marker D14Mit28. Received: 13 October 1996 / Accepted: 22 July 1997     

Genetic dissection of susceptibility to radiation-induced apoptosis of thymocytes and mapping of Rapop1, a novel susceptibility gene

Genetic dissection of susceptibility to radiation-induced apoptosis of thymocytes was performed by counting dead cells in histologically processed thymuses after 0.5 Gy of whole-body X-irradiation, using recombinant congenic (CcS/Dem) strains derived from inbred mouse strains BALB/cHeA (susceptible) and STS/A (resistant). A high (8/20) number of strains with lower dead cell scores than BALB/cHeA among CcS/ Dem recombinant congenic strains (RCS), which contain 12.5% of STS/A genome in the genetic background of BALB/cHeA strain, indicates that the difference between BALB/cHeA and STS/A is caused by several genes and that susceptibility probably requires BALB/ cHeA alleles at more than one locus. Similar results were obtained with CXS/Hg recombinant inbred (CXS/ Hg) strains. Analysis of F₂ hybrids between BALB/ cHeA and CcS-7, one of the CcS/Dem strains that showed lower dead cell scores than BALB/cHeA, demonstrated that a novel gene (Rapop1, radiation-induced apoptosis 1) controlling susceptibility to radiation-induced apoptosis in the thymus is located in the proximal region of mouse chromosome 16.     

Genetics of susceptibility to radiation-induced apoptosis in colon: two loci on Chromosomes 9 and 16

Apoptosis, a mechanism for removal of genetically damaged cells and for maintenance of desired size of cell populations, has been implicated in tumor development. Previously, we defined polymorphic loci for susceptibility to apoptosis of thymocytes Rapop1, Rapop2, and Rapop3 on mouse Chromosomes 16, 9, and 3, respectively, using recombinant congenic CcS/Dem strains, each of which contains a random set of 12.5% STS/A genome in the genetic background of BALB/cHeA. The STS/A alleles at these loci confer lower susceptibility to radiation-induced apoptosis of thymocytes than the BALB/cHeA. In the present study, we tested susceptibility of colon crypt cells to radiation-induced apoptosis. In contrast to apoptosis in thymus, the STS/A mice were more susceptible to apoptosis in colon than the BALB/cHeA. Among the CcS/Dem strains, CcS-4, CcS-7, and CcS-16 were more susceptible to apoptosis in colon than the BALB/cHeA; in thymus, the CcS-7 mice are less susceptible, and the CcS-4 and CcS-16 are not different from the BALB/cHeA. Thus, individual CcS/Dem strains showed different apoptosis susceptibility in the two organs. Analysis of (CcS-7 × BALB/cHeA)F₂ hybrids revealed linkage of susceptibility to radiation-induced apoptosis of colon crypt cells to two loci on Chrs 9 and 16, to which Rapop2 and Rapop1 are mapped. The STS/A allele at the locus on chromosome 9 results in high susceptibility to apoptosis of colon crypt cells in mice homozygous for the BALB/cHeA allele at the locus on Chr 16. Although these two loci may be identical to Rapop1 and Rapop2, they affect apoptosis in colon in a way different from that in thymus. Received: 9 October 1997 / Accepted: 29 December 1997     

Mouse lacking NAD+-linked glycerol phosphate dehydrogenase has normal pancreatic beta cell function but abnormal metabolite pattern in skeletal muscle

We surveyed the BALB/cHeA mouse, which lacks cytosolic glycerol phosphate dehydrogenase an enzyme that catalyzes a reaction in the glycerol phosphate shuttle. The other enzyme of this shuttle, mitochondrial glycerol phosphate dehydrogenase, is abundant in skeletal muscle and pancreatic islets suggesting that the shuttle's activity is high in these tissues. Levels of glycerol phosphate (low) and dihydroxyacetone phosphate (high) were very abnormal in nonislet tissue, especially in skeletal muscle. Intermediates situated before the triose phosphates in the glycolysis pathway were increased and those after the triose phosphates were generally low, depending on the tissue. The lactate/pyruvate ratio in muscle was low signifying a low cytosolic NAD/NADH ratio. This suggests that a nonfunctional glycerol phosphate shuttle caused a block in glycolysis at the step catalyzed by glyceraldehyde phosphate dehydrogenase. When exercised, mice were unable to maintain normal ATP levels in skeletal muscle. Blood glucose, serum insulin levels, and pancreatic islet mass were normal. In isolated pancreatic islets insulin release, glucose metabolism and ATP levels were normal, but lactate levels and lactate/pyruvate ratios with a glucose load were slightly abnormal. The BALB/cHeA mouse can maintain NAD/ NADH ratios sufficient to function normally under most conditions, but the redox state is not normal. Glycerol phosphate is apparently formed at a slow rate. Skeletal muscle is severely affected probably because it is dependent on the glycerol phosphate shuttle more than other tissues. It most likely utilizes glycerol phosphate rapidly and, due to the absence of glycerol kinase in muscle, is unable to rapidly form glycerol phosphate from glycerol. Glycerol kinase is also absent in the pancreatic insulin cell, but this cell's function is essentially normal probably because of redundancy of NAD(H) shuttles.     

Mutants of Escherichia coli defective in membrane phospholipid synthesis. Properties of wild type and Km defective sn-glycerol-3-phosphate acyltransferase activities.

The sn-glycerol-3-phosphate (glycerol-P) acyltransferase, the first enzyme of membrane phospholipid synthesis in Escherichia coli, was investigated in a wild type and a mutant strain defective in this activity. The mutant strain, selected as a glycerol-P auxotroph, was previously shown to contain a glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than that of its parent or revertants. The membranous mutant glycerol-P acyltransferase but did not appear to be thermolabile in vivo. Revertants no longer requiring glycerol-P for growth, showed glycerol-P acyltransferase activity with thermolability properties similar to the wild type. The second phospholipid biosynthetic enzyme, 1-acylglycerol-P acyltransferase, was not thermolabile in membranes containing a thermolabile glycerol-P acyltransferase activity. The pH optimum for the mutant acyltransferase was over 1 pH unit higher than that of the parental activity. Further, the mutant and wild type glycerol-P acyltransferase differed in their response to magnesium chloride and potassium chloride. The palmitoyl-CoA dependence of the wild type and mutant glycerol-P acyltransferase activities were different. The mutant glycerol-P acyltransferase activity was inhibited greater than 90% by Triton X-100 under conditions where the wild type activity was not affected. These experiments provide novel information about the wild type glycerol-P acyltransferase activity of E. coli and provide six additional lines of evidence for the mutant character of the glycerol-P acyltransferase in the mutant strains.     

Biosynthesis in Escherichia coli of sn-glycerol 3-phosphate, a precursor of phospholipid. Kinetic characterization of wild type and feedback-resistant forms of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Homogeneous wild type and feedback-resistant forms of the biosynthetic sn-glycerol 3-phosphate (glycerol-P) dehydrogenase of Escherichia coli (EC1.1.1.8) were subjected to two-substrate kinetic analysis. The kinetics of the NADPH-dependent reduction of dihydroxyacetone phosphate (dihydroxyacetone-P) and of the NADP-dependent oxidation of glycerol-P indicate that these reactions proceed by a sequential mechanism. Glycerol-P was a competitive inhibitor with respect to dihydroxyacetone-P for both enzymes. The wild type and feedback-resistant glycerol-P dehydrogenases had Ki values for glycerol-P of 4.4 micrometer and 43 micrometer, respectively. Therefore, the sensitivity of the wild type activity and reduced sensitivity of the feedback-resistant activity, both noted previously in crude extracts, were inherent properties of the enzymes. The patterns of product inhibition for both enzymes were identical, and the difference in the inhibition constants for glycerol-P occurred without significant alteration of any other kinetic constant determined. Kinetic mechanisms consistent with the patterns of product inhibition violated Haldane relationships and other kinetic relationships. These discrepancies suggest that glycerol-P inhibition occurs at a site distinct from the active site. The pH dependencies of the Km for dihydroxyacetone-P and the Ki for glycerol-P were markedly different suggesting the existence of an allosteric site. The addition of glycerol-P in the presence of NADPH stabilized both enzymes against thermal inactivation. Half-maximal stabilization was provided by 5 micrometer and 50 micrometer glycerol-P for the wild type and feedback-resistant enzymes, respectively. These kinetic data, considered in conjunction with previous physiologic and genetic data, indicate that the synthesis of glycerol-P is regulated in vivo by glycerol-P inhibition of the glycerol-P dehydrogenase. The data suggest that glycerol-P inhibition occurs at an allosteric, regulatory site.     

Study of anH-2K d mutant strain: C.B6-H-2 dm4

A new-H-2 mutant involving theH-2 ^d haplotype is described — C.B6-H- 2^dm4 (dm4). This mutant strain carries a gain and loss mutation which maps to theK ^d gene of theH-2 complex. Serological testing comparing the mutant and the parental BALB/cKh strain failed to detect any difference between the two strains and no antibodies could be produced, although a reciprocal mixed lymphocyte reaction was observed between mutant and parent.     

Characterization of a compensatory mutant of Leishmania major that lacks ether lipids but exhibits normal growth, and G418 and hygromycin resistance

Ether glycerolipid biosynthesis in Leishmania major initiates with the acylation of dihydroxyacetonephosphate by the glycosomal dihydroxyacetonephosphate acyltransferase LmDAT. We previously reported that a null mutant of LmDAT is severely affected in logarithmic growth, survival during stationary phase, and in virulence in mice. In addition, it lacks all ether glycerolipids, produces altered forms of the ether-lipid based virulence factors lipophosphoglycan and increased levels of GPI-anchored protein gp63. Here, we describe the characterization of a compensatory mutant of a null strain of LmDAT, Δlmdat/Δlmdat(rev). Similarly to the null mutant, the Δlmdat/Δlmdat(rev) strain formed altered forms of lipophosphoglycan and increased levels of gp63, and was avirulent in mice infection. Further, dihydroxyacetonephosphate acyltransferase activity was absent in the revertant clone, indicating that a mutation in another acyltransferase gene did not confer dihydroxyacetonephosphate specificity. In contrast, the revertant grew normally but still exhibited poor survival during stationary phase. In addition, agarose gel analysis of its genomic DNA failed to detect any amplified DNA. Surprisingly, its sensitivity to aminoglycoside based antibiotics G418 and hygromycin was lower than that of the null mutant, wild type and complemented line.     

Molecular heterogeneity for bovine maple syrup urine disease

In Poll Herefords, it is known that maple syrup urine disease results from a nonsense mutation in codon -6 of the gene for the Elα subunit of branched-chain α-keto acid dehydrogenase. The disease also occurs in Poll Shorthorns, but its molecular basis in this breed has not yet been determined. Allele-specific hybridization and allele-specific amplification, both based on the Poll Hereford mutation, failed to detect the mutant allele in Poll Shorthorn heterozygotes, and detected the normal allele in affected Poll Hereford-cross-Poll Shorthorn calves. These results demonstrate between breed molecular heterogeneity for bovine maple syrup urine disease.     

Characterization of an Escherichia coli Mutant Deficient in Dihydrolipoyl Dehydrogenase Activity

A mutant of Escherichia coli deficient in dihydrolipoyl dehydrogenase (DHL) activity has been isolated and its characteristics have been studied. The activities of the pyruvic dehydrogenase (PDC) and alpha-ketoglutaric dehydrogenase complexes (KDC) are not present in extracts of the mutant unless purified dihydrolipoyl dehydrogenase is added. Experiments with antiserum to DHL have shown that cross-reacting material exists in mutant extracts. This suggests that the dihydrolipoyl dehydrogenase mutation (dhl(-)) is a missense structural mutation. The mutation maps very close to, if not adjacent to, the ace loci, and is not linked to the suc loci. This means the dhl locus is grouped with the genes for the other components of the PDC and not with the genes for KDC. The mutation is also transducible into prototrophic strains, demonstrating that no prior mutation is necessary for the DHL activity deficiency to exist. This evidence is consistent with the idea that there is only one gene for DHL and is supported by previous biochemical studies which have shown that DHL preparations from either enzyme complex are electrophoretically and immunochemically indistinguishable. Possible mechanisms for the genetic and metabolic control of DHL, PDC, and KDC are discussed.