A complex genetic locus controlling purine nucleotide biosynthesis in yeast

Summary Mutants of the genes pur1 to pur6 excrete purine when in combination with the allele su-pur ⁺ and are resistant to growth inhibition by 8-azaadenine (8-AzAd) and 8-azaguanine (8-AzGu). In combination with su-pur, which suppresses purine excretion, pur1 and pur2 are analogue sensitive; pur3 is slightly resistant to 8-AzAd; pur4 is slightly resistant to both analogues and pur5 is completely resistant to 8-AzGu. Crosses of the pur mutants to dap, which causes sensitivity to 2,6-diaminopurine (2,6-DAP), guanine and 6-mercaptopurine (6-MP), show that dap also suppresses purine excretion and is closely linked to pur6. In combination with dap, pur1 and pur3 are analogue sensitive; pur4 is hypersensitive to guanine but resistant to 6-MP; pur5 is resistant to 2,6-DAP and guanine whilst pur2 is hypersensitive to all three compounds.The gene slw, which, like pur2, potentiates the effects of dap, also suppresses purine excretion but is not linked to any of the pur genes. The diploid slw/pur3 excretes purine.Tests for functional allelism were carried out on the closely linked genotypes su-pur ⁺, su-pur, dap, pur6, PUR6 and ade4. The results of these tests indicate that all six genotypes are functionally allelic. It is suggested that a molecular complex of the products of pur1, pur3, pur4, pur6 and slw is involved in the control of purine nucleotide biosynthesis in yeast.     

A new technique for selection of sensitive and auxotrophic mutants of E. coli: isolation of a strain sensitive to an excess of one-carbon metabolites.

Summary An E. coli strain deleted in the region malasd is used for the selection of conditional or auxotrophic mutants. Thermosensitive and auxotrophic strains have thus been isolated on plates. After selection in liquid medium, a strain has been isolated which is sensitive to excess one-carbon metabolites. It carries two mutations, smg A (near metA and argH), probably identical to rel C, and smg B (between asn and ilv), probably part of the E. coli membrane ATPase.Abbreviations dap 1 meso diaminopimelic acid - smg serine+methionine+glycine - 1:1 1 per weight     

Construction of Uracil and Tryptophan Auxotrophic Mutants from Sake Yeasts by Disruption of URA3 and TRP1 Genes

Uracil auxotrophic mutants were constructed from the sake yeasts K-701 and K-901 by successive URA3 gene disruption. First, as sake yeast is diploid, one URA3 gene was disrupted with pURA38 (AURA3 SMR1) and the heterozygous disruptant was isolated on an SM (sulfometuron methyl) plate. The other URA3 gene was disrupted with pURA36 (Δ URA3) and homozygous URA3 disruptants were isolated on FOA (5-fluoro-orotic acid) plate on which only ura3 mutants can grow. Direct URA3 gene disruption with pURA36 (Δ URA3) was also done and the uracil auxotrophic mutant was isolated. Four types of URA3 disruptants were isolated, two of which had no bacterial DNA.

A tryptophan auxotrophic mutant was constructed from one of the URA3 disruptant using pTRP14 (Δ TRP1 URA3) by gene disruption. This TRP1 disruptant was also lacking bacterial DNA.

Laboratory scale sake brewing using the auxotrophic mutants showed that these strains are very useful as recipient strains for molecular breeding of sake yeasts.     

Nucleoside auxotrophy in Drosophila: An autosomal locus yielding mutants supplementable by purine and pyrimidine ribonucleosides

Summary Two allelic auxotrophic mutants at a locus close to the bw locus (2–104.5) of Drosophila melanogaster are described. The mutants respond to dietary ribonucleosides (uridine, cytidine, adenosine, guanosine and inosine) but less well to bases or pyrimidine precursors. This phenotype is unique to these mutants.We suggest that the mutants are defective in phosphoribosyl pyrophosphate biosynthesis.     

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.     

Using adaptive mutagenesis system for identification of early genes encoding de novo purine biosynthesis in methylotrophic yeast <Emphasis Type="Italic">Pichia methanolica</Emphasis> MH4

A collection of spontaneous “Roman’s mutants” (1654 mutants) for early genes of purine biosynthesis PUR1–PUR5 was obtained from 16 parental ade1 (pur6) and ade2(pur7) strains of the methylotrophic yeast Pichia methanolica. Two genes, bifunctiional ADE7,4(PUR2,5) and ADE5(PUR4), were identified earlier. For identification of the two remaining early genes (ADE3 and ADE8), a novel approach was used: a comparison of spectra of spontaneous Roman’s mutants and relative sizes of genes (with regard to the length of polypeptides in amino acid residues). Significant correlation between relative sizes of genes and a proportion of mutants in the spectrum was shown in yeast Saccharomyces cerevisiae (according to analysis of data from the literature).     

Three purine auxotrophic loci on the second chromosome of Drosophila melanogaster

Mutations at three second-chromosomal loci of Drosophila melanogaster have been isolated, mapped, and shown to be purine nucleoside auxotrophs. Two of the loci, adenosine2 and adenosine3, located at map positions 18.4 and 20, respectively, produce mutations which are supplementable with adenine, adenosine, and inosine. Guanosine supplements mutations at the burgundy locus (55.7); this locus was described previously through a pteridine eye-color defect but identified as an auxotrophic locus after the isolation of a new allele, bur ^gua2-1 . The mutation ade2-1 also has defective pteridine metabolism.This work was supported by NSERC Grant A3269 to D. Nash and by an AHFMR graduate studentship to M. E. Johnstone.     

Ertosterol biosynthesis in Saccharomyces cerevisiae: mutants deficient in the early steps of the pathway.

Summary Thermosensitive mutants, auxotrophic for ergosterol synthesis, have been isolated, analyzed genetically and their enzymatic deficiencies investigated. These mutants were classified into seven unlinked complementation groups. These groupes lack the following enzymatic activities: squalene epoxidase (erg 1), 2,3-oxidosqualene-lanosterol cyclase (erg 7), phosphomevalonic kinase (erg 8), mevalonic kinase (erg 12) and squalene synthetase (erg 9, erg 10, erg 11).     

Genetic analysis of methylotrophic yeastCandida boidinii PLD1

This paper reports the initial experiments for genetic analysis of the haploid methylotrophic yeastCandida boidinii PLD1. The collection of multiply marked auxotrophic mutants was obtained after treatment with UV-light or X-rays. Protoplasts from several mutants were fused by the PEG-CA²⁺ technique and five prototrophic hybrids were isolated. The genetic structure of the hybrids was studied by means of spontaneous and induced mitotic segregation. Our data suggest that hybrids are diploids, heterozygous by parental auxotrophic markers. We obtained genetic linkage between mutationslys2-8-met-3 from one hand andade-17-arg-24 from the other. The genetic maps constructed showed similar characteristics concerning both the order of the markers and their map distances.     

Mu-induced rifamycin-resistant mutations not located in the rpoB gene of Escherichia coli

Summary A new class of rifamicin-resistant mutants of Escherichia coli was obtained by lysogenic insertions of bacteriophage Mu Amp DNA. Rifamycin resistance is closely linked to the ampicillin resistance conferred by the prophage. Mapping by conjugation with auxotrophic markers revealed that the rifamycin-resistant mutations are located between 28 and 37 min on the E. coli chromosome standard map, some distance from the rpoB gene at 89.5 min. The DNA-dependent RNA polymerase of these mutants is highly sensitive to rifampicin.     

Chromosomal location of gene governing the trehalose utilization in Escherichia coli K12.

Summary Several mutants unable to utilize trehalose were isolated from Escherichia coli. Their genetic analysis led to determine the following gene order on the chromosomal map: pur B-dad R-tre-hem A-trp. Furthermore, the tre gene belongs to the inversion of the trp chromosomal region between E. coli and S. typhimurium.     

Genetic control of Escherichia coli K-12 strains' assimilation of 2,6-diaminopurine as a purine source]

Mutations of the resistance to 2,6-diaminopurine (apt), which affect adenine phosphoribosyltransferase, fail to permit the growth of Escherichia coli pur mutants (purine auxotrophs which cannot make inosine monophosphate de novo) on the medium with 2,6-diaminopurine (DAP) as the sole source of purines. Addition of a small amount of hypoxantine, but not guanine, stimulated the growth of mutants of pur apt and pur apt+ genotypes on the medium with DAP. The utilization of DAP as purine source in the presence of hypoxantine is blocked by mutations guaC (guanosine monophosphate reductase), add (adenosine deaminase) and pup (purine necleoside phosphorylase), suggesting that DAP are utilized via purine nucleoside phosphorylase and adenosine deaminase. The drm mutation (that increases the level of pentose-1-phosphate in the cell) does not activate the utilization of DAP. The results indicate that a step, that limits the utilization of DAP as the sole source of purines by pur mutants of E. coli, is the deamination of DAP nucleoside.     

Construction of a Quadruple Auxotrophic Mutant of an Industrial Polyploid Saccharomyces cerevisiae Strain by Using RNA-Guided Cas9 Nuclease

Industrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain, Saccharomyces cerevisiae ATCC 4124, with ura3, trp1, leu2, and his3 auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNA-guided Cas9 nuclease now enable metabolic engineering of polyploid S. cerevisiae strains that have been widely used in the wine, beer, and fermentation industries.     

Mapping of the chromosome ofMycobacterium phlei by means of mutagenesis of the replication point

The aim of the present work was to construct a replication map of the chromosome ofMycobacterium phlei. The method of mutagenesis of the replication point by means of nitrosoguanidine was applied to synchronously multiplying populations. Back mutations and forward mutations were induced m auxotrophic mutants PAmet and PAleu as well as in double auxotrophic mutants with methlomne as the reference marker and the following order of replication of eleven genes on the chromosome was thus established:leu-Eth, Res-Stm, Oyk-pur-met, arg, Cyk-Bac-inl     

Production of lysine by mutants of escherichia coli K 12 in a medium with lactose

In an effort to use whey for lysine production, we isolated from a β-galactosidase-hyperproducing strain of E. coli K 12 multiple mutants – auxotrophic, regulatory and penicillin-resistant. These mutants exhibited for the most part a high reversion rate but some of them produced about 2 mg/ml lysine in an enriched fermentation medium.     

Selektion Actidion-resistenter Mutanten bei Neurospora crassa sowie ihre genetische und biochemische Analyse

Summary Conidia of an actidione-sensitive wildtype strain of Neurospora crassa were irradiated with UV-light. They were then plated into nutrient-agar, either with or without actidione. The latter plates were incubated for several hours, before nutrient agar containing actidone was layered onto the plates. Colonies formed in both sets of plates were isolated as actidione-resistent. They were studied further by genetic and biochemical means.Pre-incubation of the irradiated conidia before subjecting them to the action of actidione increased the mutant yield considerably, as compared to immediate plating with the drug. E.g. a 13 hours pre-incubation gave ca. 100 times more resistent colonies than were obtained without pre-incubation (Fig. 2). Their resistent phenotype was stable on vegetative propagation.17 mutants were mapped by crossing them with suitable tester-strains. Of them, 14 were found to belong to linkage group I, the remaining to linkage group V. The mutants are, therefore, considered as characterizing resp. genes act-1 and -2 of Hsu (1963). Act-1 and -2 mutants were crossed with suitable auxotrophic strains to obtain auxotrophic, actidione-resistent isolates. These were combined on minimal medium with auxotrophic, actodione-sensitive strains of the same mating type. Conidia of the arising heterokaryotic mycelia were tested on minimal medium with and without actidione. In these tests resistence of act-1 and -2 mutants was found to be dominant over the sensitivity of the wildtype. However, an analysis of nuclear ratios in the conidial populations by differential plating does not exclude incomplete dominance of act-1.Incorporation of 14-C-leucine into protein of conidia of the wildtype was strongly inhibited by 1 actidione/ml. Resistence in two mutants, representing the two separate genes, was accompanied by a marked decrease of this inhibition. No significant differences in the amount of inhibition were found between the two mutants. It is suggested that cytoplasmic ribosomes may be the cellular components influenced by actidione. In the case of the mutant cells the actidione is no longer effective in this capacity, possibly because of changes in the ribosomal proteins.     

Direct mating between diploid sake strains of Saccharomyces cerevisiae

Various auxotrophic mutants of diploid heterothallic Japanese sake strains of Saccharomyces cerevisiae were utilized for selecting mating-competent diploid isolates. The auxotrophic mutants were exposed to ultraviolet (UV) irradiation and crossed with laboratory haploid tester strains carrying complementary auxotrophic markers. Zygotes were then selected on minimal medium. Sake strains exhibiting a MATa or MATα mating type were easily obtained at high frequency without prior sporulation, suggesting that the UV irradiation induced homozygosity at the MAT locus. Flow cytometric analysis of a hybrid showed a twofold higher DNA content than the sake diploid parent, consistent with tetraploidy. By crossing strains of opposite mating type in all possible combinations, a number of hybrids were constructed. Hybrids formed in crosses between traditional sake strains and between a natural nonhaploid isolate and traditional sake strains displayed equivalent fermentation ability without any apparent defects and produced comparable or improved sake. Isolation of mating-competent auxotrophic mutants directly from industrial yeast strains allows crossbreeding to construct polyploids suitable for industrial use without dependence on sporulation.     

The induction of mutants in a non-acid-fast strain ofMycobacterium phlei with nitrous acid

The inactivation and mutagenic effets of nitrous acid on a non-acid-fast strain ofMycobacterium phlei were studied. It was found that 0.017m NaNO₂ at pH 4.4 may be used for the induction of auxotrophic mutants, scotochromogenic and achromogenic mutants and STM-resistant mutants. Three doubly auxotrophic mutants, three mutants requiring amino acids and three mutants depending on vitamins were obtained. One mutant was not classified. Eighteen scotochromogenic mutants were isolated, seventeen of them were orange. Only ten achromogenic mutants were isolated. Twelve scotochromogenic and eight achromogenic mutants could be used in further genetic studies as they did not revert spontaneously to photochromogeny. Six auxotrophic mutants could be used due to their low frequency of spontaneous reversions. The frequency of STM-resistant mutants increased on an average seven-fold after the mutagenic treatment as compared with the spontaneous frequency.     

Reproduction of bacteriophage T6 and T7 in auxotrophic mutants ofE. coli B

Summary Phage T₆ and T₇ did not reproduce in mutants ofE. coli auxotrophic for the amino acids methionine, leucine, serine, proline, histidine, tyrosine, phenylalanine and tryptophane unless these amino acids were present in the medium. Very small amounts of these auxotrophic factors (0.01 γ/ml) are able to support phage multiplication. Much care has to be taken to avoid impurities in the medium and to deplete the endogenous reserves of the host cells.     

Evidence for the parasexual cycle in a strain of aspergillus flavus containing virus-like particles

Eight isolates of A. flavus and A. parasiticus were screened for the presence of virus-like-particles (VLP). Qnly A.flavus strain NRRL 5565 contained detectable VLP. Spore color and auxotrophic mutants were induced in this strain and evidence for the parasexual cycle was obtained. Attempts to form heterokaryons between 3 auxotrophs of the VLP-containing strain and 9 auxotrophs from two different aflatoxigenic strains were unsuccessful.