Cloning and expression inEscherichia coli of the sucrase gene fromBacillus subtilis

Summary A recombinant cosmid carrying the sucrase gene (sacA) was obtained from a colony bank ofE. coli harboring recombinant cosmids representative of theB. subtilis genome. It was shown that thesacA gene is located in a 2 kbEcoRI fragment and that the cloned sequence is homologous to the corresponding chromosomal DNA fragment. A fragment of 2 kb containing the gene was subcloned in both orientations in the bifunctional vector pHV33 and expression was further looked for inB. subtilis andE. coli. Complementation of asacA mutation was observed in Rec⁺ and Rec^- strains ofB. subtilis. Expression of sucrase was also demonstrated inE.coli, which is normally devoid of this activity, by SDS-polyacrylamide gel electrophoresis, specific immunoprecipitation and assay of the enzyme in crude extracts. The specific activity of the enzyme depended on the orientation of the inserted fragment. The saccharolytic activity was found to be cryptic inE. coli since the presence of the recombinant plasmids did not allow the transport of [U¹⁴C] sucrose and the growth of the cells.It was shown also that the recombinant cosmid contained part of the neighboring locus (sacP) which corresponds to a component of the PEP-dependent phosphotransferase system of sucrose transport ofB. subtilis.     

Chromosomal location of mutations affecting sucrose metabolism in Bacillus subtilis Marburg

Summary Mutations affecting sucrose metabolism have been mapped by PBS1 transduction on the Bacillus subtilis chromosome in seven loci sacA, sacB, sacQ, sacR, sacS, sacT and sacU. sacA and sacB are presumed to be the structural genes of a sucrase and a levansucrase respectively. sacR, sacS and sacT correspond to groups of mutations leading to constitutive synthesis of sucrase or both sucrase and levansucrase. In sacQ, sacS and sacU are located either mutations increasing the level of synthesis of levansucrase specifically (sacQ ^h , sacS ^h , sacU ^h ) or mutations abolishing specifically the synthesis of levansucrase (sacU ^– ). sacA, sacS and sacT map to the left of purA16. sacQ is located to the left of thr5, sacB and sacR between cysB3 and hisA1 and sacU between uvr1 and gtaB.     

Map locations of some mutations conferring resistance to arginine hydroxamate in Bacillus subtilis 168

Summary Mutations conferring resistance to arginine hydroxamate in Bacillus subtilis 168 have been located on the genetic map by PBSl-mediated transduction. The majority of these mutations, belonging to classes 1, 2 and 4 of Harwood and Baumberg (1977) and affecting only expression of arginine catabolic enzymes, map at a locus designated ahrA cotransducible with cysA, purA and sacA. The order of markers in this region appears to be sacA-ahrA-purA-cysA. Certain anomalies were observed in the properties of Pur⁺ transductants from crosses with an Ahr donor and a purA recipient. A single ahr mutation (class 3), also affecting only arginine catabolism, maps between ctrA and sacA at a locus designated ahrB. Two others (class 6), affecting simultaneously enzymes of both arginine biosynthesis and catabolism, map between lys and aroD at a locus designated ahrC. Preliminary attempts to define the nature of functional products specified by these ahr loci suggest that a protein is encoded at ahrA.Abbreviations OAT ornithine aminotransferase - OCT ornithine carbamoyltransferase - AS argininosuccinase - AH arginine hydroxamate - Ahs, Ahr arginine hydroxamate-sensitive or-resistant - Ahos, Ahor sensitive or resistant to arginine hydroxamate in the presence of ornithine - ahr allele conferring arginine hydroxamate resistance - Out⁺, Out^- able or unable to utilise ornithine as sole nitrogen source     

Genetics of oxidative phosphorylation: mitochondrial loci determining ossamycin-, venturicidin- and oligomycin-resistance in yeast

Summary With a view towards identifying new ATPase loci on the mitochondrial genome a large number of oligomycin-, ossamycin- and venturicidin-resistant mutants were isolated after MnCl₂ mutagenesis. The mutants were subjected to mass-screens which divided them into different cross-resistance phenotype-classes and also distinguished the common OLI1 mutations from the mutations at all other loci.Allelism tests between examples of the different classes of phenotype indicated that the majority of mutations in the population mapped at the previously known loci OLI1, OLI2, OLI3 and OLI4. Mutations conferring specific ossamycin resistance defined two new loci, namely OSS1 and OSS2 which are linked to the OLI2 and OLI1 loci respectively. A few rare mutations comprise a new locus OLI5 which is linked to the OLI1 locus (12.6% total recombination).In conclusion we can now say that there are two unlinked segments of the mitochondrial genome, each of which is composed of several distinct, genetically-linked loci. One segment contains the OLI1, OLI3, OLI5 and OSS2 loci and the other the OLI2, OLI4 and OSS1 loci. The phenotypically-distinguishable mutations described herein should facilitate fine-structure mapping of these two segments.     

Molecular Structure of the Lactobacillus plantarum Sucrose Utilization Locus: Comparison with Pediococcus pentosaceus

Structure of the sucrose utilization locus in a Lactobacillus plantarum type strain was studied using PCR and Southern hybridization. Restriction map analysis revealed its high similarity to the sequenced sucrose utilization locus of Pediococcus pentosaceus pSRQ1. The L. plantarum locus proved to contain oppositely oriented scrA and the scrBRagl operon, but not agaS. The L. plantarum sucrase gene (scrB) was partly sequenced. A higher (98.6%) homology was revealed between scrB than between the 16S rRNA genes of L. plantarum and P. pentosaceus, suggesting horizontal transfer of the sucrose utilization locus between the genera of lactic acid bacteria. Amino acid sequence analysis showed that the ScrB proteins of the two species belong to a subfamily of glycosyl hydrolase family GH32 which includes various -fructosidases.     

Genetic studies of mutations at two loci of Drosophila melanogaster which cause a wide variety of homeotic transformations

Summary The ash-1 locus is in the proximal region of the left arm of the third chromosome of Drosophila melanogaster and the ash-2 locus is in the distal region of the right arm of the third chromosome. Mutations at either locus can cause homeotic transformations of the antenna to leg, proboscis to leg and/or antenna, dorsal prothorax to wing, first and third leg to second leg, haltere to wing, and genitalia to leg and/or antenna. Mutations at the ash-1 locus cause, in addition, transformations of the posterior wing and second leg to anterior wing and second leg, respectively. A similar spectrum of transformations is caused by mutations at yet another third chromosome locus, trithorax. One extraordinary aspect of mutations at all three of these loci is that they cause such a wide variety of transformations. For mutations at both of the loci that we have studied the expression of the homeotic phenotype is both disc-autonomous (as shown by injecting mutant discs into metamorphosing larvae) and cell autonomous (as shown by somatic recombination analysis). The original mutations which identified these two loci, although lethal, manifest variable expressivity and incomplete penetrance of the homeotic phenotype suggesting that they are hypomorphic. The phenotype of double mutants which were synthesized by combining different pairs of those original mutations manifest for two of the four pairs a greater degree of expressivity and slightly more penetrance of the homeotic transformations. This mutual enhancement suggests that the products of both loci interact in the same process. A third double mutant expresses a discless phenotype.Additional alleles have been recovered at both the ash-1 and the ash-2 loci. Some of these alleles as homozygotes or transheterozygotes express the wide range of transformations revealed first by double mutants. One of the alleles at the ash-1 locus when homozygous and several transheterozygous pairs can cause either the homeotic transformation of discs or the absence of those discs. The fact that these two defects, absence of specific discs and homeotic transformations of those same discs can be caused by mutations within a single gene suggests that the activity of the product of this gene is essential for normal imaginal disc cell proliferation. Loss of that activity leads to the absence of discs, whereas, reduction of that activity leads to homeotic transformations.     

Ribosomal subunits affected by antibiotic resistance mutations at seven chloroplast loci in Chlamydomonas reinhardtii

Summary Mutations at seven recombinationally distinct chloroplast loci confer antibiotic resistance on chloroplast ribosomes of the green alga Chlamydomonas reinhardtii. Assays of polynucleotide-directed amino acid incorporation by ribosomes reconstituted from mutant and wild type subunits demonstrate that streptomycin, neamine/kanamycin and spectinomycin resistance mutations specifically affect the small ribosomal subunit, whereas mutations to erythromycin resistance affect the large subunit. Although in each case the subunit site of antibiotic resistance is the same as that observed in analogous mutations in Escherichia coli, the number of loci conferring resistance to a given antibiotic differs in the two organisms. We have previously shown that streptomycin resistance mutations in Chlamydomonas map at five discrete loci (one nuclear and four chloroplast), and that mutations to neamine/kanamycin and spectinomycin resistance appear to define a single chloroplast locus. Results presented here confirm our previous report that all chloroplast erythromycin resistance mutations isolated to date fall into two recombinationally distinct loci, and indicate that mutants at one of these loci may be further divided on the basis of their level of cross resistance to other macrolide antibiotics.     

Characterization of a novel low molecular weight sucrase from filamentous fungus Termitomyces clypeatus

An extracellular sucrase from the culture filtrate of filamentous basidiomycota Termitomyces clypeatus grown on high sucrose (5%, w/v) was purified by gel filtration chromatography, ion exchange chromatography and HPGPLC. The biochemical properties, molecular weight and conformation of sucrase produced were significantly different from the sucrase earlier purified from sucrose (1%, w/v) medium in the fungus. Purified sucrase was characterized as a low molecular weight protein of 13.5 kDa as approximated by SDS-PAGE and HPGPLC and exhibited predominantly random coil conformation in far-UV CD spectra. The enzyme was optimally active at 47 °C and pH 5.0. K_m and catalytic activity of the enzyme for sucrose were found to be 3.5 mM and 1.06 U/mg/mM, respectively. The enzyme was maximally active towards sucrose than to raffinose and sucrase activity was significantly inhibited by bivalent metal ions and reducing group agents. The results indicated that due to changes in aggregation pattern, molecular organization of purified sucrase, produced in high sucrose medium, was altered and was different from the previously reported enzyme. This is the first report of a sucrase of such low size showing activity.     

N-Ethyl-N-nitrosourea-induced prenatally lethal mutations define at least two complementation groups within the embryonic ectoderm development (eed) locus in mouse Chromosome 7

Two loci [l(7)5Rn and l(7)6Rn] defined by N-ethyl-N-nitrosourea (ENU)-induced, prenatally lethal mutations were mapped by means of trans complementation crosses to mice carrying lethal deletions of the albino (c) locus in Chromosome (Chr) 7. Both loci were found to map to the subregion of the Mod-2-sh-1 interval that contains the eed (embryonic ectoderm development) locus. eed has been defined by the inability of embryos homozygous for certain c deletions to develop beyond the early stages of gastrulation. Evidence for at least two loci necessary for normal prenatal development, rather than one locus, that map within the eed interval came from the observation that two prenatally lethal mutations, 3354SB [l(7)5Rn ^3354SB ] and 4234SB [l(7)6Rn ^4234SB ], could complement each other in trans, but could not each be complemented individually by c deletions known to include the eed locus. A somewhat leaky allele of l(7)5Rn [l(7)5Rn ^1989SB ] was also recovered, in which hemizygotes are often stillborn and homozygotes exhibit variable fitness and survival. The mapping of the loci defined by these mutations is likely to be useful for genetic, molecular, and phenotypic characterization of the eed region, and mutations at either locus (or both loci) may contribute to the eed phenotype.     

S-adenosyl methionine requiring mutants in Saccharomyces cerevisiae: evidences for the existence of two methionine adenosyl transferases.

Summary Mutants requiring S-adenosyl methionine (SAM) for growth have been selected in Saccharomyces cerevisiae. Two classes of mutants have been found. One class corresponds to the simultaneous occurrence of mutations at two unlinked loci SAM1 and SAM2 and presents a strict SAM requirement for growth on any medium. The second class corresponds to special single mutations in the gene SAM2 which lead to a residual growth on minimal medium but to normal growth on SAM supplemented medium or on a complex medium like YPGA not containing any SAM. These genetic data can be taken as an indication that Saccharomyces cerevisiae possesses two isoenzymatic methionine adenosyl transferases (MAT). In addition, SAM1 and SAM2 loci have been identified respectively with the ETH-10 and ETH2 loci previously described.Biochemical evidences corroborate the genetic results. Two MAT activities can be dissociated in a wild type extract (MATI and MATII) by DEAE cellulose chromatography. Mutations at the SAM1 locus lead to the absence or to the modification of MATII whereas mutations at the SAM2 locus lead to the absence or to the modification of MATI. Moreover, some of our results seem to show that MATI and MATII are associated in vivo.     

Genetic determination of ubiquinol-cytochromec reductase

Summary In order to find new genetic loci on the yeast mitochondrial DNA, especially mutations affecting the structure and function of ubiquinol-cytochrome c reductase, 45 independently arisen mutants resistant to mucidin have been isolated after MnCl₂ mutagenesis. The majority of the mutants exhibited increased sensitivity to chloramphenicol, diuron and antimycin A, respectively. it was shown by several criteria that all mutants resulted from mutations localized on the mitochondrial DNA.The allelism tests revealed that these mutations fall into three distinct loci muc1, muc2 and muc3. Mutations at a new locus muc3 were correlated with the changes in the binding or inhibitory sites on the inner mitochondrial membrane. Multifactorial crosses involving the mucidin resistance mutations and mitochondrial mutations conferring resistance to chloramphenicol, erythromycin, oligomycin and diuron revealed that the studied mutations at the loci muc1, muc2 and muc3 did not significantly influence the process of mitochondrial recombination and its control by the mitochondrial locus . The locus muc1 was found to be allelic to the locus diu2. The locus muc2 which was found to be allelic to cob1 locus appears to be linked to the locus oli1 but unlinked to the loci , cap1, ery1 and muc1. The new locus muc3 appears to be weakly linked to the locus diu1 but unlinked to the loci , cap1, ery1, oli1 and muc1.The results are consistent with the gene order oli1-muc2-muc3-diu1-muc1-oli2 and suggest the participation of at least three mucidin resistance loci and one diuron resistance locus in the biogenesis of the bc ₁ complex of the mitochondrial respiratory chain.     

Genetic instability at the cut locus of Drosophila melanogaster induced by the MR-h12 chromosome

Summary Unstable mutations were generated at the cut locus by the MR-h12 factor which induces male recombination. The unstable allele ct ^MR2, containing the MR-transposon in the cut locus is a very powerful mutator producing a number of different viable and lethal mutations both in the cut locus and outside it.I describe several types of mutations: stable reversion to wild type, which were sometimes associated with the appearance of unstable mutations in other loci; of stable deficiencies at the cut locus (lethals); new unstable mutations at different loci with the ct ^MR2 allele conserved; new unstable cut alleles with a phenotype other than that of ct ^MR2. The possible mechanisms of these mutational events are discussed. The genetic system constructed in the present work affords an opportunity for molecular studies of the cut locus and the MR-transposon, as a sequence from the cut locus has recently been cloned (Tchurikov et al. 1981).     

Identification and characterization of NF1-related loci on human chromosomes 22, 14 and 2

Neurofibromatosis type 1 (NF1) is a frequent hereditary disorder. The disease is characterized by a very high mutation rate (up to 1/10000 gametes per generation). NF1-related loci in the human genome have been implicated in the high mutation rate by hypothesizing that these carry disease-causing mutations, which can be transferred to the functional NF1 gene on chromosome arm 17q by interchromosomal gene conversion. To test this hypothesis, we want to identify and characterize the NF1-related loci in the human genome. In this study, we have localized an NF1-related locus in the most centromeric region of the long arm of chromosome 22. We demonstrate that this locus contains sequences homologous to cDNAs that include the GAP-related domain of the functional NF1 gene. However, the GAP-related domain itself is not represented in this locus. In addition, cosmids specific to this locus reveal, by in situ hybridization, NF1-related loci in the pericentromeric region of chromosome arm 14q and in chromosomal band 2q21. These cosmids will enable us to determine whether identified disease-causing mutations are present at the chromosome 22-associated NF1-related locus. Received: 18 December 1995 / Revised: 5 February 1996     

Sorbose resistant mutants of Aspergillus nidulans

Summary Mutations to L-sorbose resistance in Aspergillus nidulans have been characterised at two loci. At one locus (sorA) mutations confer cross resistance to 2-deoxy-D-glucose and result in a defect in sugar uptake. At the other locus (sorB) sorbose resistance results from loss of phosphoglucomutase and is accompanied by pronounced morphological abnormality but not by loss of ability to utilise D-galactose.     

Genes affecting the major outer membrane proteins of Escherichia coli K-12: Mutations at nmpA and nmpB

Summary Strains of Escherichia coli K-12 carrying mutations at either of two distinct loci (nmpA and nmpB) produce a new outer membrane pore protein which is not present in wild type cells. Mutations at either of these loci result in sensitivity to phage TC45, which can use this new protein as its receptor, and the new protein (the NmpAB protein) appears to be identical in both NmpA and NmpB mutants. In order to determine whether both of these loci contain structural genes for the NmpAB protein, strains carrying secondary mutations at either of these loci which produced altered proteins were sought by selecting for resistance to phage TC45. Mutants which produced proteins with altered electrophoretic mobility and altered peptide maps were isolated from strains carrying both nmpA and nmpB mutations, and these secondary mutations mapped at the same sites as the original mutations leading to production of the NmpAB protein. This suggests that both loci contain structural genes. Strains mutant at nmpB which can no longer produce the protein can mutate at the nmpA locus to produce the new protein, indicating that both genes can exist in the same cell. Since the altered proteins of mutant strains could be distinguished from one another, we attempted to construct strains in which both nmpA and nmpB were expressed. In all cases only the protein produced by the nmpB mutation was produced, indicating some form of cooperative regulation of the two genes.     

The intracellular sucrase (SacA) of Zymomonas mobilis is not involved in sucrose assimilation

The intracellular sucrase SacA from Zymomonas mobilis was purified to homogeneity from a recombinant E. coli strain containing the SacA gene under an expression system. The protein was monomeric with a molecular mass of 58 kDa. The sucrase activity was maximal at 25 °C and thermal stability of the purified protein was low (50% recovery after 30 min at 46 °C ). The activation energy was low at 33 kJ mol^–1. Maximum activity was at pH 6.5. Activity was strongly inhibited (>99%) by SH blocking reagents and reducing agents slightly (10–60%) increased the activity of purified SacA. The sucrase showed a low K _M (42 mM) and k _cat (125 s^–1) which indicated its very low efficiency for sucrose hydrolysis. A mutant strain of Z. mobilis not able to grow on sucrose was isolated. This strain (ZM4S) lacked the two sucrases SacB and SacC but SacA was present in the intracellular fraction. Therefore, SacA alone is unable to allow growth Z. mobilis on sucrose.     

Isolation of polymorphic microsatellite markers for Begonia sutherlandii Hook. f.

Seven polymorphic microsatellite loci have been characterized for investigating population structure in the patchily distributed herb Begonia sutherlandii. Two loci (BSU3 and BSU4) exhibited population specific null alleles; primer redesign and allele sequencing for one of these loci showed two transition mutations in the original primer site. Two loci exhibited imperfect repeat polymorphisms due to single base pair indels in the flanking region (locus BSU6) and in the microsatellite region itself (BSU7). Transversion mutations were also found in the microsatellite region of locus BSU7. The remaining three loci amplified in all individuals tested and appeared to conform to a simple stepwise mutation pattern.     

A genetic and physiological analysis of late flowering mutants in Arabidopsis thaliana

Summary Monogenic mutants of the early ecotype Landsberg erecta were selected on the basis of late flowering under long day (LD) conditions after treatment with ethyl methanesulphonate or irradiation. In addition to later flowering the number of rosette and cauline leaves is proportionally higher in all mutants, although the correlation coefficient between the two parameters is not the same for all genotypes. Forty-two independently induced mutants were found to represent mutations at 11 loci. The mutations were either recessive, intermediate (co locus) or almost completely dominant (fwa locus). The loci are located at distinct positions on four of the five Arabidopsis chromosomes. Recombinants carrying mutations at different loci flower later than or as late as the later parental mutant. This distinction led to the assignment of eight of the loci to three epistatic groups. In wild type, vernalization promotes flowering to a small extent. For mutants at the loci fca, fve, fy and fpa, vernalization has a large effect both under LD and short day (SD) conditions, whereas co, gi, fd and fwa mutants are almost completely insensitive to this treatment. SD induces later flowering except for mutants at the co and gi loci, which flower with the same number of leaves under LD and SD conditions. This differential response of the mutants to environmental factors and their subdivision into epistatic groups is discussed in relation to a causal model for floral initiation in Arabidopsis thaliana.     

The genetics of a putative social trait in natural populations of yeast

The sharing of secreted invertase by yeast cells is a well‐established laboratory model for cooperation, but the only evidence that such cooperation occurs in nature is that the SUC loci, which encode invertase, vary in number and functionality. Genotypes that do not produce invertase can act as ‘cheats’ in laboratory experiments, growing on the glucose that is released when invertase producers, or ‘cooperators’, digest sucrose. However, genetic variation for invertase production might instead be explained by adaptation of different populations to different local availabilities of sucrose, the substrate for invertase. Here we find that 110 wild yeast strains isolated from natural habitats, and all contained a single SUC locus and produced invertase; none were ‘cheats’. The only genetic variants we found were three strains isolated instead from sucrose‐rich nectar, which produced higher levels of invertase from three additional SUC loci at their subtelomeres. We argue that the pattern of SUC gene variation is better explained by local adaptation than by social conflict.     

Delayed fermentation of sucrose by certain haploid species ofSaccharomyces

Summary Several haploid species ofSaccharomyces andSchiz. octosporus were shown to ferment sucrose in Durham tubes after a delay of 3 to 4 weeks. Detailed studies were done with a strain ofS. rouxii. The delayed fermentation of sucrose was not caused by mutationselection or by inducible enzyme formation, since young glucose grown cells after drying, freezing, aging or autolysis contained an active sucrase. Cells pretreated by drying or freezing fermented sucrose nearly as fast as glucose. After autolysis, the sucrase ofS. rouxii is only present in the cell debris and not in the autolysate. The use of a heavy inoculum in the van Iterson-Kluyver fermentometer resulted in a slow, but non-delayed fermentation. Variation in the pH or sucrose concentration had little effect on the delayed fermentation. It is suggested that after sufficient aging of the cells, the cell wall permeability undergoes a rather abrupt change, allowing the sucrose to come in contact with the sucrase of the cells.