Agonist induction, conformational selection, and mutant receptors

Current models of receptor activation are based on either of two basic mechanisms: agonist induction or conformational selection. The importance of one pathway relative to the other is controversial. In this article, the impossibility of distinguishing between the two mechanisms under a thermodynamic approach is shown. The effect of receptor mutation on the constants governing ligand-receptor equilibria is discussed. The two-state model of agonism both in its original formulation (one cycle) and including multiple active states (multiple cycles) is used. Pharmacological equations for the double (two cycles) two-state model are derived. The simulations performed suggest that the double two-state model of agonism can be a useful model for assessing quantitatively the changes in pharmacological activity following receptor mutation.     

Respiratory-deficient,yeastlike mutant of Mucor

Stable mutants of Mucor bacilliformis having lost the ability to grow filamentously and to sporulate occur spontaneously with a frequency of about one in every 3000 colonies. On solid and in liquid medium these mutants have a typical yeastlike morphology and reproduce by budding. The detailed study of one of these mutants shows that the inability to form filaments and spores is accompanied by the loss of cytochrome oxidase activity. This mutant is unable to take up oxygen but has a high level of alcoholic fermentation, which appears to be the major if not the sole source of energy.This investigation was supported by Public Health Service Grant AI 07804 05.     

A new mouse mutant, skijumper

Low blood sugar levels are a well-known cause of severe illness and often death in newborn humans, especially those that are small for age. Few of the causes of neonatal hypoglycemia are known, and many remain to be found. We describe a novel mouse mutant, skijumper (skimp), in which pups, despite feeding well, have low levels of glucose and develop opisthotonos, followed by death typically within a few days after birth. Genetic mapping studies have localized the lesion to a approximately 1 cM interval on mouse Chromosome (Chr) 7 between D7Mit318 and D7Mit93. We have carried out extensive analysis to define the phenotype and its likely cause. In addition to low blood glucose, affected skijumper mice have lowglycogen and ketone levels. Mass spectrometric analysis of blood samples has excluded major defects in amino acid metabolism. Initial biochemical analyses suggested a defect in ketogenesis as one possible cause of this phenotype. However, measurements of levels and activities of carnitine, carnitine palmitoyl transferases, and other enzymes involved in ketogenesis, along with studies of mitochondrial structure and function, did not demonstrate significant differences between skijumper, unaffected littermates, and control wild-type mice. These results indicate that abnormal enzyme activity in known pathways does not appear to be the primary biochemical lesion in skijumper. The skijumper may be a new valuable model for studying and understanding one type of neonatal morbidity and death.     

Renal cortical and medullary blood flow responses to L-NAME and ANG II in wild-type, nNOS null mutant, and eNOS null mutant mice

Experiments in wild-type (WT; C57BL/6J) mice, endothelial nitric oxide synthase null mutant [eNOS(-/-)] mice, and neuronal NOS null mutant [nNOS(-/-)] mice were performed to determine which NOS isoform regulates renal cortical and medullary blood flow under basal conditions and during the infusion of ANG II. Inhibition of NOS with N(omega)-nitro-l-arginine methyl ester (l-NAME; 50 mg/kg iv) in Inactin-anesthetized WT and nNOS(-/-) mice increased arterial blood pressure by 28-31 mmHg and significantly decreased blood flow in the renal cortex (18-24%) and the renal medulla (13-18%). In contrast, blood pressure and renal cortical and medullary blood flow were unaltered after l-NAME administration to eNOS(-/-) mice, indicating that NO derived from eNOS regulates baseline vascular resistance in mice. In subsequent experiments, intravenous ANG II (20 ng x kg(-1) x min(-1)) significantly decreased renal cortical blood flow (by 15-25%) in WT, eNOS(-/-), nNOS(-/-), and WT mice treated with l-NAME. The infusion of ANG II, however, led to a significant increase in medullary blood flow (12-15%) in WT and eNOS(-/-) mice. The increase in medullary blood flow following ANG II infusion was not observed in nNOS(-/-) mice, in WT or eNOS(-/-) mice pretreated with l-NAME, or in WT mice administered the nNOS inhibitor 5-(1-imino-3-butenyl)-l-ornithine (1 mg x kg(-1) x h(-1)). These data demonstrate that NO from eNOS regulates baseline blood flow in the mouse renal cortex and medulla, while NO produced by nNOS mediates an increase in medullary blood flow in response to ANG II.     

Azotobacter vinelandii ferredoxin I: cloning, sequencing, and mutant analysis

The structure of Azotobacter vinelandii ferredoxin I (AvFdI) has been extensively characterized by a variety of techniques. Although its physiological function is unknown, it has long been implicated as being involved in electron donation to nitrogenase. Here we report that the AvFdI gene (fdxA) has been cloned from an EcoRI digest lambda library using a synthetic oligonucleotide probe and that its sequence has been determined. The amino acid sequence deduced from the DNA sequence is identical to the previously published protein sequence. Analysis of the promoter region indicates that AvFdI is not a nif specific gene product. A mutant of A. vinelandii has been constructed which is identical to the wild-type, at the DNA level, except that the fdxA gene has been interrupted by insertion of a kanamycin cartridge. This mutant, called LM100, does not synthesize AvFdI but does synthesize the Fe and MoFe proteins of nitrogenase and grows at wild-type rates under N2-fixing conditions. This demonstrates that AvFdI is not required for N2 fixation by A. vinelandii. There is a small acidic protein, which is present in wild-type A. vinelandii, whose level is dramatically increased in LM100. The nature of this protein is under further investigation.     

Craniofacial, caudal, and visceral anomalies associated with mutant sirenomelic mice

Craniofacial anomalies were correlated with mutant murine sirenomelia. Ninety-eight newborn sirens from heterozygous matings were examined and analyzed. In the 96 sirens that had intact craniofacial structures, micrognathia was seen in 39% of the sirens, microstomia in 34%, macroglossia in 26%, and cleft palate in 21%. Even when not cleft, the siren palates were narrower and more highly arched than those of nonsiren littermates. The frequency of abnormal craniofacial development was greater in those sirens that were more severely affected caudally. Even though some earlier studies had indicated a preponderance of males, 46 of the 95 sirens with intact pelvic viscera were females. Fifty-three percent of the sirens were monopodal, 35% were apodal, and 11% were dipodal. A penile-like projection on the genital tubercle occurred on 15 apodal sirens and four monopodal sirens; all but three of these sirens were males. Bladder agenesis was seen in 100% of the sirens, anal atresia in 80%, and bilateral renal agenesis in 43%. No siren was found with bilaterally normal kidneys. The srn gene responsible for sirenomelia might either directly affect the embryo at both the caudal and cranial regions or indirectly affect the embryo by producing lateral mechanical compression at both these sites. The srn gene was earlier characterized as autosomal-recessive; our data confirm this. Sirenomelia was found in only 11% of the newborns from crosses of carrier mice in the colony. Analyses of uterine contents at days 12-14 suggest that the srn gene is fully penetrant, but often lethal, during the fetal period.     

shibire, a neurogenic mutant of Drosophila

Embryos of the temperature-sensitive mutant shibirets 1 were given short exposures to the restrictive temperature during the stage when neuroblasts segregate from the presumptive epidermis. The resulting lethal phenotype, expansion of the nervous system at the expense of the epidermis, is characteristic of a group of mutants called neurogenic mutants. Exposures as short as 20 min were sufficient to promote the neurogenic phenotype. Cell masses from heat-pulsed embryos could be cultured in vivo as tumorous masses which retained some characteristics of neural tissue. An examination of the neurogenic region from heat-pulsed embryos revealed numerous packets of extracellular vesicles and coated pits blocked in endocytosis.     

Carbon isotope ratios of amylose,amylopectin and mutant starches

Carbon isotope ratios (expressed as δ¹³C values) were determined for various sources of starch and the starch fractions amylose and amylopectin. The δ¹³C values of amylose were consistently less negative, 0.4–2.3 ‰, than those of amylopectin in kernal starch from maize (Zea mays) and barley (Hordeum vulgare) and in tuber starch from potato (Solanum tuberosum). Kernel starch isolated from the maize mutants wx1 and ae1, with known genetic lesions in the starch biosynthetic pathway, also showed significant differences in δ¹³C values. Collectively, these results suggest that variation in carbon isotope ratios in the amylose and amylopectin components of starch may be attributed to isotopic discrimination by the enzymes involved in starch biosynthesis.     

Characterization of a new simian virus 40 mutant, tsA3900, isolated from deletion mutant tsA1499.

The simian virus 40 (SV40) mutant tsA1499 contains an 81-base-pair deletion in the region of A gene encoding the C-terminal portion of the large T antigen. This mutant is particularly interesting, since it is a temperature-sensitive mutant that is apparently able to separate the lytic growth and transforming functions of the SV40 large T antigen at 38.5 degrees C. We report the isolation of a tsA1499 revertant (tsA1499-Rev) which is no longer temperature sensitive for lytic growth but still contains the 81-base-pair deletion of tsA1499. Marker rescue experiments with tsA1499-Rev or wild-type strain 830 (wt830) DNAs revealed that the original tsA1499 mutant contained a second mutation within the HindIII-Fnu4HI restriction fragment between 0.425 and 0.484 map units. Sequencing of this DNA fragment from the tsA1499, tsA1499-Rev, and wt830 viruses revealed that tsA1499 contained a single-base transversion (C to G) at 0.455 map units (nucleotide 4261). This transversion resulted in the creation of a new RsaI cleavage site in the tsA1499 DNA and predicts an arginine-to-threonine substitution at amino acid position 186 in the mutant large T antigen. The DNA sequence of the tsA1499-Rev HindIII-Fnu4HI fragment was identical to that of wt830. To determine whether tsA1499 was temperature sensitive for lytic growth solely as a result of the newly discovered point mutation or because of a combination of the point and deletion mutations, a series of viruses were constructed which contained the point mutation, the deletion mutation, both mutations, or neither. This was done by ligating the PstI A and B DNA fragments from either tsA1499 or wt830 and transfecting the ligated DNA into BSC-1H monkey kidney cells. This experiment revealed that all viruses containing the point mutation (the tsA1499 PstI A DNA fragment) were temperature sensitive for lytic growth, regardless of the presence of the 81-base-pair deletion (the tsA1499 PstI B DNA fragment). This newly discovered point mutation, at nucleotide 4261, is therefore unique, since to our knowledge it is the first tsA mutation to be described in the 0.455-map-unit region of the SV40 genome. We then tested the effect of this unique mutation on the ability of the SV40 virus to transform cultured rat cells to anchorage independence.(ABSTRACT TRUNCATED AT 400 WORDS)     

Physiological aspects of nm-g mutant: An ecdysteroid-deficient mutant of the silkworm,Bombyx mori

We examined physiological and endocrinological properties of the nm-g mutant of the silkworm, whose development was arrested at 1st or 2nd larval instar. The larva of the nm-g homozygote continued to feed and grow past the stage when a normal larva moulted to the next instar. The nm-g larva attained more than twice the body weight of a normal moulting larva, but remained in the same instar with no sign of the moulting until it finally died. The ecdysteroid concentration in the haemolymph of the nm-g larva was reduced and an increase in the titre, which occurred before moulting in the normal larva, was not observed. Injection of 20-hydroxyecdysone induced the nm-g larva to moult and ecdyse to the next larval instar. From these results, we conclude that nm-g is an ecdysteroid-deficient mutant.     

Spontaneous alternation, motor activity, and spatial learning in hot-foot mutant mice

Hot-foot mutant mice, characterized by defective innervation of Purkinje cells and an ataxic gait, were less active than normal mice in a T-maze. In spontaneous alternation testing with either single or multiple trials, hot-foot mutants, contrary to normal mice, did not alternate above chance. Moreover, the mutants had a higher number of errors and higher escape latencies in a water-filled Z-maze. These results indicate that in addition to motor coordination deficits, these cerebellar mutants have deficits in spatial learning and perseverate choices of maze arms.     

Isolation,characterization, and mapping of the stay green mutant in rice

Leaf color turns yellow during senescence due to the degradation of chlorophylls and photosynthetic proteins. A stay green mutant was isolated from the glutinous japonica rice Hwacheong-wx through N-methyl-N-nitrosourea mutagenesis. Leaves of the mutant remained green, while turning yellow in those of the wild-type rice during senescence. The stay green phenotype was controlled by a single recessive nuclear gene, tentatively symbolized as sgr(t). All the phenotypic characteristics of the mutant were the same as those of the wild-type lines except for the stay green trait. The leaf chlorophyll concentration of the mutant was similar to that of the wild-type before heading, but decreased steeply in the wild-type during grain filling, while very slowly in the mutant. However, no difference in photosynthetic activity was observed between the stay green mutant and the yellowing wild-type leaves, indicating that senescence is proceeding normally in the mutant leaves and that the mutation affects the rate of chlorophyll degradation during the leaf senescence. Using phenotypic and molecular markers, we mapped the sgr(t) locus to the long arm of chromosome 9 between RFLP markers RG662 and C985 at 1.8- and 2.1-cM intervals, respectively. Received: 29 April 2001 / Accepted: 17 July 2001     

DNA damage, superoxide, and mutant K-ras in human lung adenocarcinoma cells

DNA single-strand breaks (quantitative comet assay) were assessed to indicate ongoing genetic instability in a panel of human lung adenocarcinoma cell lines. Of these, 19/20 showed more DNA damage than a nontransformed cell line from human peripheral lung epithelium, HPL1D. DNA damage was significantly greater in those derived from pleural effusates vs those from lymph node metastases. DNA strand breaks correlated positively with superoxide (nitroblue tetrazolium reduction assay), and negatively with amount of OGG1, a repair enzyme for oxidative DNA damage. Levels of CuZn superoxide dismutase varied moderately among the lines and did not correlate with other parameters. A role for mutant K-ras through generation of reactive oxygen species was examined. Cells with mutant K-ras had significantly lower amounts of manganese superoxide dismutase (MnSOD) vs those with wild-type K-ras, but MnSOD protein correlated positively with superoxide levels. In a subset of cell lines with similar levels of MnSOD, comparable to those in HPL1D cells, K-ras activity correlated positively with levels of both superoxide and DNA strand breaks. These results suggest that persistent DNA damage in some lung adenocarcinoma cells may be caused by superoxide resulting from mutant K-ras activity, and that OGG1 is important for prevention of this damage.     

Co-accumulation of prephenate, L-arogenate, and spiro-arogenate in a mutant of Neurospora

A mutant strain of Neurospora crassa blocked in each of the initial steps of tryptophan, tyrosine, and phenylalanine biosynthesis was previously shown to accumulate and secrete prephenate and L-arogenate (Jensen, R.A., Zamir, L.O., St. Pierre, M., Patel, N., and Pierson, D.L. (1977) J. Bacteriol. 132, 896-903). We now report the co-accumulation of yet another compound which was identified (Zamir, L.O., Tiberio, R., Jung, E., and Jensen, R.A. (1982) J. Biol. Chem. (1983) 258, 6486-6491) as the lactam derivative of L-arogenate. This structure, spiro-arogenate, undergoes a facile acid-catalyzed conversion to L-phenylalanine (as does L-arogenate). Since L-arogenate is conveniently quantitated as 5-dimethylaminonapthalene-1-sulfonyl (dansyl)-phenylalanine following acidification and dansylation, the unknown presence of spiro-arogenate may easily lead to overestimation of L-arogenate present in mixtures. Reliable quantitative assays for both L-arogenate and spiro-arogenate in mixtures were designed utilizing [3H]dansyl-chloride and exploiting the inability of the spiro-arogenate molecule to be dansylated in contrast to L-arogenate. The initial appearance of spiro-arogenate during accumulation lagged behind prephenate and L-arogenate, and spiro-arogenate accumulation leveled off after 5 days while prephenate and L-arogenate accumulations continued. It seems likely that spiro-arogenate is derived directly from L-arogenate. Prephenate, L-arogenate, and spiro-arogenate comprised about 70, 15, and 15% fractions of the total accumulation in a representative accumulation experiment designed to maximize spiro-arogenate yields. Modest variations in co-metabolite ratios were obtained under nutritional conditions where carbon source, growth temperature, duration of incubation time, and amino acid additions were experimental variables.     

Streptomycin-resistant, asporogenous mutant of Bacillus subtilis.

Summary Mutants of Saccharomyces cerevisiae lacking pyruvate kinase (EC 2.7.1.40) are described. These have less than 0.5% of the pyruvate kinase activity of the wild type. All the other glycolytic enymes are present in normal amounts in these mutants. The mutation is recessive and segregates in diploids as a single gene. Five alleles examined fail to complement one another. Tetrad analysis and mitotic recombination data place the mutation on the left arm of chromosome I distal to cys 1. The majority of single-step spontaneous revertants on glucose regain the enzyme activity fully and this activity appears, by a number of criteria, to be due to the same enzyme present in the wild type. Some of these revertants become nuclear petites. The mutants do neither grow on nor ferment sugars but do grow on ethyl alcohol or pyruvate. Glucose addition to cultures growing on alcohol arrests growth until glucose is exhausted. The steady state rate of glucose utilization is slower than in the wild type. This is associated with the accumulation of as much as 5 moles P-enolpyruvate per g wet weight of cells and proportional amounts of 2-P-glyceric and 3-P glyceric acids.The mutation is believed to involve some regulatory element in the synthesis of pyruvate kinase.     

A slow-growing, ribosomal mutant of Salmonella typhimurium

Summary The mechanisms of S. typhimurium reversion from histidine dependence (his ^–) to histidine independence (his ⁺) were studied. Genetic and phenotypic characteristics of revertants induced by nitrosoguanidine were analyzed. Among them a class of slow-growing revertants was selected. It is found that all of these slow-growing revertants carry the original UGA nonsense mutation within the histidine operon. They are streptomycin sensitive and no specific suppressor(s) for UGA nonsense codon are demonstrable. The suppression takes place in the absence of conventional nonsense UGA suppressor(s). It is seemingly due to a ribosomal mutation which in turn is likely to produce ambiguity in the process of translation and which suppresses the UGA nonsense codon. The rate of both in vivo and in vitro protein synthesis is significantly reduced. The fact that streptomycin, at sublethal doses, reduced the growth rate of these mutants, probably because of the simultaneous burden of two ambiguity factors, suggests that the mutants described may be regarded as a kind of ram (ribosomal ambiguity) mutants with a his ^– sup ^–genotype. Their capacity to translate poly-U is reduced and in that respect they differ from ram mutants of Escherichia coli.