Prototrophic Regulatory Mutants of Adenylosuccinate Synthetase in Yeast

MUTANTS of the genes ade12 and ade13 in Saccharomyces cerevisiae require adenine, which suggests that they are defective in the conversion of inosine monophosphate (IMP) to adenosine monophosphate (AMP). Two enzymes are required for this conversion: adenylosuccinate lyase and adenylosuccinate synthetase. The former has been shown to be absent in mutants of ade13 (ref. 1) so that one can infer that ade12 specifies the latter. Mutants of ade12 have been isolated on the basis of an adenine insensitive pigment accumulation by strains carrying ade1 or ade2 (refs. 1 and 2). They seem to synthesize purines constitutively and excrete hypoxanthine¹. These observations suggest that in addition to its catalytic function adenylosuccinate synthetase has an important function in the regulation of purine synthesis.     

Molecular cloning and characterization of a novel muscle adenylosuccinate synthetase,AdSSL1, from human bone marrow stromal cells

Vertebrates have muscle and non-muscle isozymes of adenylosuccinate synthetase (AdSS, EC 6.3.4.4), which catalyzes the first committed step in AMP synthesis. A novel muscle isozyme of adenylosuccinate synthetase, human AdSSL1, is identified from human bone marrow stromal cells. AdSSL1 is 98% identical to mouse muscle type AdSS1 and contains conserved sequence and structural features of adenylosuccinate synthetase. Human AdSSL1 gene is mapped to chromosome 14p32.33. After stimulation, leukemia cells express AdSSL1 in a time-dependent manner different from that of non-muscle adenylosuccinate synthetase. The human AdSSL1 is predominantly expressed in skeletal muscle and cardiac tissue consistent with the potential role for the enzyme in muscle metabolism. Overexpressed AdSSL1 protein in COS-7 cells locates in cytoplasm. Recombinant AdSSL1 protein possesses typical enzymatic activity to catalyze adenylosuccinate formation. The identification of human AdSSL1 with predominant expression in muscle tissue will facilitate future genetic and biochemical analysis of the enzyme in muscle physiology. (Mol Cell Biochem 269: 85–94, 2005)     

Nitrate and nitrite reductase negative mutants of N2-fixingAzospirillum spp.

Chlorate resistant spontaneous mutants ofAzospirillum spp. (syn.Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants fromA. brasilense and 13 fromA. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr^–). Most of the mutants were also nitrite reductase negative (nir^–), only 3 remaining nir⁺. Two mutants from nr⁺ nir⁺ parent strains lost only nir and became like the nr⁺ nir^– parent strain ofA. brasilense. No parent strain or nr⁺ mutant showed any nitrogenase activity with 10 mM NO ₃ ^– . In all nr^– mutants, nitrogenase was unaffected by 10 mM NO ₃ ^– . Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir^–. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr^– nir^–) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.     

The spontaneous azaguanine-resistant mutants of diploid human fibroblasts

Summary The range of incidences of azaguanine-resistant colonies in cultures of fibroblasts from 16 unrelated humans was 0.4×10^-6 to 19×10^-6 and the mean value was 4.1×10^-6. A fluctuation test showed that most or all of the mutant colonies derived from mutations that occurred during in vitro proliferation of the fibroblasts and before exposure to azaguanine. The estimated rate of spontaneous mutation was 0.45×10^-6 to 1.8×10^-6 per cell generation. At least ten independent mutants, comprising two general classes, were studied. Class I mutants were a minority and resembled cells from boys having the Lesch-Nyhan syndrome: they had very little HG-PRT activity, showed maximum resistance to azaguanine and could not utilize hypoxanthine for growth. At least 90% of the mutants were in Class II: their apparent HG-PRT activities ranged between normal and Lesch-Nyhan amounts, they were partially sensitive to azaguanine and they could utilize hypoxanthine. Some Class II mutants resembled cells cultured from a family having an X-chromosomal mutant gene that does not cause the Lesch-Nyhan syndrome but does confer resistance to azaguanine, although the quantity of HG-PRT activity is apparently normal and hypoxanthine can be utilized. Electrophoretic differences between the HG-PRT activities of normal and mutant strains were not detected but other qualitative alterations were observed in some mutants.Paper No. 1558 from the Laboratory of Genetics.Supported by N.I.H. Grants GM-06983 and GM-15422 and by a grant from the Food Research Institute of The University of Wisconsin, Madison, Wisconsin.Supported by Grant He 753-1 from Die Deutsche Forschungsgemeinschaft.     

Selection and properties of spontaneous mutants ofListeria monocytogenes ATCC 15313 resistant to different bacteriocins produced by lactic acid bacteria strains

The frequency of spontaneous mutants ofListeria monocytogenes ATTC 15313 resistant to the inhibitory action of three bacteriocins of lactic acid bacteria previously discovered in our laboratory (mesenterocin 52, curvaticin 13, and plantaricin C19) was estimated to be in the range of 10^–3 to 10^–4. The phenotypic character of resistance was stable during several generations in the absence of contact with bacteriocins. The resistance was not due to the inactivation of bacteriocins nor to a modification of their adsorption on the target cells. The selected mutants resistant to one of the bacteriocin cited above showed a cross-resistance to the two other bacteriocins, but not to nisin.     

The product of theade1 gene inSchizosaccharomyces pombe: A bifunctional enzyme catalysing two distinct steps in purine biosynthesis

Summary The assignment of the knownade genes to steps in purine biosynthesis inSchizosaccharomyces pombe has been completed with the demonstration that anade3 mutants lacks FGAR amidotransferase,ade1A mutants lack GAR synthetase andade1B mutants lack AIR synthetase. A comparison of enzyme activity with map position forade1 mutants shows that (1) complementingade1A mutants lack GAR synthetase but possess wild type amounts of AIR synthetase, (2) complementingade1B mutants lack AIR synthetase but posses variable amounts of GAR synthetase, (3) non-complementing mutants lack both activities. In wild type strains the two activities fractionate together throughout a hundred-fold purification. Hence theade1 gene appears to code for a bifunctional enzyme catalysing two distinct steps in purine biosynthesis. The two activities are catalysed by two different regions of the polypeptide chain which can be altered independendently by mutation. Gel filtration studies on partially purified enzymes from wild type and various complementing mutant strains, indicate that the bifunctional enzyme is a multimer consisting of between four and six sub-units of 40,000 daltons each. GAR synthetase activity is associated with both the monomeric and multimeric forms but AIR synthetase is only associated with the multimer. A comparison of enzyme levels between diploids and their original complementing haploid strains suggests that complementation is due to hybrid enzyme formation.     

Metabolism of purine bases, nucleosides and alkaloids in theobromine-forming Theobroma cacao leaves

We examined the purine alkaloid content and purine metabolism in cacao (Theobroma cacao L.) plant leaves at various ages: young small leaves (stage I), developing intermediate size leaves (stage II), fully developed leaves (stage III) from flush shoots, and aged leaves (stage IV) from 1-year-old shoots. The major purine alkaloid in stage I leaves was theobromine (4.5 μmol g^–1 fresh weight), followed by caffeine (0.75 μmol g^–1 fresh weight). More than 75% of purine alkaloids disappeared with subsequent leaf development (stages II–IV). In stage I leaves, ¹⁴C-labelled adenine, adenosine, guanine, guanosine, hypoxanthine and inosine were converted to salvage products (nucleotides and nucleic acids), to degradation products (ureides and CO₂) and to purine alkaloids (3- and 7-methylxanthine, 7-methylxanthosine and theobromine). In contrast, ¹⁴C-labelled xanthine and xanthosine were not used for nucleotide synthesis. They were completely degraded, but nearly 20% of [8-¹⁴C]Xanthosine was converted in stage I leaves to purine alkaloids. These observations are consistent with the following biosynthetic pathways for theobromine: (a) AMP → IMP → 5′-xanthosine monophosphate → xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine; (b) GMP → guanosine → xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine; (c) xanthine → 3-methylxanthine → theobromine. Although no caffeine biosynthesis from ¹⁴C-labelled purine bases and nucleosides was observed during 18 h incubations, exogenously supplied [8-¹⁴C]Theobromine was converted to caffeine in young leaves. Conversion of theobromine to caffeine may, therefore, be slow in cacao leaves. No purine alkaloid synthesis was observed in the subsequent growth stages (stages II–IV). Significant degradation of purine alkaloids was found in leaves of stages II and III, in which [8-¹⁴C]Theobromine was degraded to CO₂ via 3-methylxanthine, xanthine and allantoic acid. [8-¹⁴C]Caffeine was catabolised to CO₂ via theophylline (1,3-dimethylxanthine) or theobromine.     

Characterisation of new symbiotic Medicago truncatula (Gaertn.) mutants, and phenotypic or genotypic complementary information on previously described mutants

From a pool of Medicago truncatula mutants—obtained by gamma-irradiation or ethyl methanesulfonate mutagenesis—impaired in symbiosis with the N-fixing bacterium Sinorhizobium meliloti, new mutants are described and genetically analysed, and for already reported mutants, complementary data are given on their phenotypic and genetic analysis. Phenotypic data relate to nodulation and mycorrhizal phenotypes. Among the five new mutants, three were classified as [Nod⁺ Fix^– Myc⁺] and the mutations were ascribed to two loci, Mtsym20 (TRV43, TRV54) and Mtsym21 (TRV49). For the two other new mutants, one was classified as [Nod^–/+ Myc⁺] with a mutation ascribed to gene Mtsym15 (TRV48), and the other as [Nod^– Myc^-/+] with a mutation ascribed to gene Mtsym16 (TRV58). Genetic analysis of three previously described mutants has shown that [Nod^–/+ Myc⁺] TR74 mutant can be ascribed to gene Mtsym14, and that [Nod^–/+ Myc^–/+] TR89 and TRV9 mutants are ascribed to gene Mtsym2 (dmi2). Using a detailed analysis of mycorrhizal phenotype, we have observed a delayed typical arbuscular mycorrhizal formation on some mutants that present thick lens-shaped appressoria. This phenotype was called [Myc^–/+] and mutants TR25, TR26, TR89, TRV9, P1 and Y6 were reclassified as [Myc^–/+]. Mutant P1 was reclassified as [Nod^–/+] because of a late nodulation observed on roots of this mutant.     

L-Lysine production by mutants of Bacillus licheniformis

Summary A bacterium able to grow at 46°C was isolated from soil and identified asBacillus licheniformis. L-Lysine producing mutants were derived from the bacterium by the introduction of resistance to S-(2-amino)-ethylcysteine (thialysine) and auxotrophy.One of the mutants, HBR-2 (Thialysine^r, Leu^–, Homoserine^–), produced L-lysine at a concentration of 30 mg/ml in a molasses medium containing 10% reducing sugar.     

Analysis of sorbose resistance in Neurospora crassa in heterokaryons of sorbose resistant mutants; contribution to the genetics of active transport

Summary Suitable auxotrophic markers were introduced into sorbose-resistant mutants and the sorbose-sensitive wildtype strain. Pairwise combinations of one resistant and one sensitive strain each as well as of two sensitive strains were then grown on minimal-agar to obtain forced heterocaryons. The growth behaviour of these on minimal-agar with and without added sorbose was compared.Of seven resistant mutants, representing six separate genes, among which were genes A and B, six mutants were recessive to the wildtype. The seventh, representing gene C, was recessive only with regard to colony-size, but intermediate with regard to germination counts. Heterocaryons forced between pairs of 2 closely linked mutants (intragenic case of the type A^– ₁+A^– ₂) were resistant, as were the separate mutants. However two heterocaryons forced between pairs of unlinked mutants (intergenic case of the type A^–+B^–) were sorbose sensitive. Heterocaryons forced between A or B-mutants and the C-mutant mentioned, unlinked to either A or B (intergenic cases of the type A^–+C^– and B^–+C^–) were more sensitive than the separate mutants but more resistant than the wildtype.It follows that sorbose-resistant mutants in heterocaryons of the intergenic types can complement each others defects (no growth complementation), but can not do so in heterocaryons of the intragenic type. Their complementation is considered to be the result of the activity of the intact wildtype genes homologous to the defective ones that are contained together in the multinucleate cells of the heterocaryons. This complementation may be taken as evidence for the recessiveness resp. intermediate expression of the different resistant mutants.Since none of the mutants checked so far were dominant compared to the wildtype, none of them can be a regulator-mutant. The possibility of explaining them as suppressor mutants is restricted by their recessiveness to mechanisms of suppression giving a recessive phenotype. An alternative explanation suggests that the respective wildtype genes may contain structural information for the synthesis of permeases involved in sorbose transport. The mutants would then be resistant due to defective permeases. Their recessiveness is in full accord with this suggestion.

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II. Teil einer Habilitationsschrift bei der Naturwissenschaftlichen Fakultät der Universität München.     

Adenylosuccinate Synthetase and Adenylosuccinate Lyase Deficiencies Trigger Growth and Infectivity Deficits in Leishmania donovani

Leishmania are auxotrophic for purines, and consequently purine acquisition from the host is a requisite nutritional function for the parasite. Both adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL) have been identified as vital components of purine salvage in Leishmania donovani, and therefore Δadss and Δasl null mutants were constructed to test this hypothesis. Unlike wild type L. donovani, Δadss and Δasl parasites in culture exhibited a profoundly restricted growth phenotype in which the only permissive growth conditions were a 6-aminopurine source in the presence of 2′-deoxycoformycin, an inhibitor of adenine aminohydrolase activity. Although both knock-outs showed a diminished capacity to infect murine peritoneal macrophages, only the Δasl null mutant was profoundly incapacitated in its ability to infect mice. The enormous discrepancy in parasite loads observed in livers and spleens from mice infected with either Δadss or Δasl parasites can be explained by selective accumulation of adenylosuccinate in the Δasl knock-out and consequent starvation for guanylate nucleotides. Genetic complementation of a Δasl lesion in Escherichia coli implied that the L. donovani ASL could also recognize 5-aminoimidazole-(N-succinylocarboxamide) ribotide as a substrate, and purified recombinant ASL displayed an apparent K_m of ∼24 μm for adenylosuccinate. Unlike many components of the purine salvage pathway of L. donovani, both ASL and ADSS are cytosolic enzymes. Overall, these data underscore the paramount importance of ASL to purine salvage by both life cycle stages of L. donovani and authenticate ASL as a potential drug target in Leishmania.     

Toxicity and mutagenicity of X-rays and [I]dUrd or [H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

Isolation of thymidine kinase-deficient rat hepatoma cells by selection with bromodeoxyuridine,Hoechst 33258, and visible light

The photosensitivity of bromodeoxyuridine (BrdU)-substituted cells is known to be markedly enhanced by the fluorochrome Hoechst 33258. Since the incorporation of BrdU into nucleic acids depends upon its prior phosphorylation via thymidine kinase (TK; EC 2.7.1.21), cells deficient in TK activity are refractory to photoinduced killing. These observations suggested that combined treatment with BrdU, Hoechst 33258, and visible light would constitute an efficient selective strategy for the recovery of TK^– mutant cells. In this report we describe a single-step selection protocol which reduced the survival of TK⁺ cells by a factor of 10⁵ without affecting the viability of TK^– mutants. This procedure was used to isolate H4IIEC3-derived rat hepatoma cells deficient in TK activity. The properties of several TK^– hepatoma clones are discussed.The valuable technical assistance of J. M. Courvall, F. R. Parker, and M. M. Smith is acknowledged. These studies were supported by grant GM26449 from the National Institutes of Health. A.M.K. was supported by a postdoctoral fellowship from the American Cancer Society, California Division. T.G.L. is a Leukemia Society of America postdoctoral fellow. R.E.K.F. is the recipient of an American Cancer Society Faculty Research Award.     

Drosophila purine auxotrophy: New alleles ofadenosine2 exhibiting a complex visible phenotype

New mutant alleles of theadenosine2 locus (ade2; 2–17.7) have been isolated using the eye-color phenotype exhibited by the prototype auxotrophic alleleade2 ¹ as the screening criterion. The new mutants form a single complementation group, suggesting that they all exhibit purine auxotrophy and defective formylglycineamide ribotide amidotransferase enzyme, likeade2 ¹. Tests carried out on particular new alleles confirm these suggestions. The new mutants all exhibit more extreme physical defects than the prototype. They have wing abnormalities like mutants defective in pyrimidine biosynthesis and reduced bristles like those defective in protein synthesis; thus they exhibit the combined visible phenotype ofrudimentary wings,rosy eyes, andbobbed bristles. Cytogenetic analysis places the locus in the interband proximal to26B1-2.This work was supported by NSERC Operating Grant A3269 to D.N., an Alberta Heritage Foundation for Medical Research Postdoctoral Fellowship to S.Y.K.T., and National Institute on Aging Grant AG00029 to D.P.     

Specificity of the generation and expression of enhanced anti-plasmacytoma immunity by spleen cells from melphalan-treated MOPC-315 tumor bearers

Summary We have shown previously that low-dose melphalan (L-PAM) therapy of mice bearing a large MOPC-315 plasmacytoma enables their hitherto immunosuppressed spleen cells to exert potent anti-MOPC-315 cytotoxicity following in vitro immunization with MOPC-315 tumor cells [3]. Here we show that, following in vitro immunization with MOPC-315 tumor cells, spleen cells from such L-PAM-treated MOPC-315 tumor bearers exhibited enhanced T-cell-mediated cytotoxicity not only against the MOPC-315 tumor, but also against another plasmacytoma (MOPC-104E) possessing surface immunoglobulin (SIg) of a different idiotype than the MOPC-315 cells, as well as against a variant of the MOPC-315 tumor which does not produce nor possess SIg (SIg^–MOPC-315). The enhanced cytotoxicity was directed against target antigens which are not expressed on the surface of the syngeneic WEHI 22.1 thymoma or the natural killer-sensitive YAC-1 cells. Plasmacytoma shared antigens, other than immunoglobulins, were able to stimulate spleen cells from L-PAM-cured MOPC-315 tumor bearers to generate in vitro a secondary type anti-plasmacytoma cytotoxic response. L-PAM-cured MOPC-315 tumor bearers exhibited in vivo immunity against SIg^–MOPC-315 tumor cells, which was sufficiently triggered by the SIg^– cells to bring about the rejection of a challenge of at least 100-fold the minimal lethal tumor dose of the SIg^–MOPC-315 cells. Thus, SIg^– MOPC-315 tumor cells present among SIg⁺ tumor cells in the parental MOPC-315 tumor inoculum [9, 26] can be eradicated in the L-PAM-treated MOPC-315 tumor bearers by the immune response to SIg⁺ tumor cells as well as by the immune response to SIg^– tumor cells themselves.Supported by Research Grant CA-35761 from the National Cancer Institute and Research Grant IM-435 from the American Cancer SocietyIn partial fulfillment of the requirements for the Doctor of Philosophy degree.     

Control of feedback repression of the enzymes of purine biosynthesis in Aerobacter aerogenes

Studies have been made of the regulation of the synthesis of six purine biosynthetic enzymes: P-ribosyl-PP amidotransferase (I), P-ribosyl glycinamide synthetase (II), P-ribosyl formyl glycinamide amidotransferase (IV), adenylosuccinate lyase (VIII-IIA), adenylosuccinate synthetase (IA), and IMP dehydrogenase (IG). Wild type Aerobacter aerogenes and two purine requiring mutants derived from it, were grown with limiting or excess adenine or guanine, cell extracts prepared, and enzyme activities measured.     

Suppression of temperature sensitive mutants of bacteriophage T4 by bacterial opal suppressors

Summary Some of the partial revertants from opal (UGA) mutants of bacteriophage T4 are temperature sensitive in su ^– host cells but are still temperature resistant in su ⁺ cells. Hence these revertants are missense mutants suppressible by bacterial opal suppressors. Such a suppression may be explained in terms of codon-anticodon interactions by the wobble hypothesis.     

Action of thiamine on auditory evoked potentials in the guinea pig

A technique is proposed for quantifying the effects of physiologically active substances at the periphery of the auditory analyzer. It was found that applying 1×10^–11 to 1×10^–3 M thiamine to the membrane of guinea pig cochlear round window (fenestra rotunda) produces a rise in the amplitude and a reduction in the latency of the N₁ and N₂ components of auditory nerve action potentials, waves I and II of brainstem auditory evoked potentials occurring in response to an acoustic stimulus. It is suggested that this effect is produced by facilitated synaptic transmission at synapses between hair cells and spiral ganglia neurons under the action of thiamine penetrating into the cochlea.A. V. Palladin Institute of Biochemistry, Academy of Sciences of the Ukrainian SSR, Kiev. A. I. Kolomiichenko Research Institute of Otolaryngology, Ministry of Public Health of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 654–660, September–October, 1986.     

Pleotropic mutants from Alcaligenes eutrophus defective in the metabolism of hydrogen,nitrate, urea,and fumarate

Chromosomal mutants of Alcaligenes eutrophus unable to grow with molecular hydrogen as the energy source also failed to grow with nitrate as the terminal electron acceptor or as a nitrogen source. The mutants (Hno^–) (i) formed neither soluble nor particulate hydrogenase antigens, (ii) expressed only about 50% the wild type level of ribulosebisphosphate carboxylase activity, and (iii) transported nickel, an essential constituent of active hydrogenase, at a significantly lower rate than wild type cells. Moreover, the mutants grew very slowly with urea as nitrogen source and did not express urease. Growth on formamide was also affected and formamidase activity was induced to only a very low level. Growth of the Hno^– mutants on succinate, glutamate, fumarate, and malate was significantly slower than wild type, and a reduced rate of succinate incorporation into the mutant cells was demonstrated. The highly pleiotropic phenotype of Hno^– mutants is indicative of a chromosomal gene with a considerable physiological importance. It affected the expression of both chromosomal and megaplasmid encoded systems of energy, carbon, and nitrogen metabolism. Thus, the hno mutation restricts the metabolic versatility but does not affect the basic metabolic functions of the organism.     

Isolation and characterization of two asporogenous rifampicin resistant mutants of B. thuringiensis

Two rifampicin resistant mutants, Rif^r12 and Rif^r15, of B. thuringiensis Berliner have been isolated and characterized as true asporogenous mutants. Cells of Rif^r12 were blocked between stage I and stage II in the sporulation sequence; whereas cells of Rif^r15 mutant were blocked between stage II and stage III. It has been shown that two active forms of RNA-polymerase were present at t5 in Rif^r15 cells; as for wild type strain, the subunit composition of form I and form II were respectively ββ'_mα2 and β′βα2. In Rif^r12 cells, only one enzymatic form was found at t5 ; the subunit composition was determined as β′βσ_mα2 ; such a composition was characteristic for sporulation enzyme of wild type strain at t1,5. It is concluded that the sigma modification which occurs at about t1, is anterior to the β′ modification which is closely correlated with the forespore septum completion (t2). Thus, the timing of the modifications of B. thuringiensis RNA-polymerase previously suggested was clearly confirmed through the present study.In addition, both mutants present reduced levels of intracellular proteolytic activities, as compared with wild type strain, and the role of proteases is discussed.