Functional domains of theEscherichia coli ferric uptake regulator protein (Fur)

The functions of N- and C-terminal domains of the Fur repressor ofEscherichia coli in promoter recognition and dimerization were studied. We investigated the ability of fusion proteins containing the N- or C-terminal domain of Fur to dimerize and to repress a Fur-regulatedlacZ fusion gene. The N-terminal domain, when fused to the C-terminal domain of the repressor C1857, repressed a Fur-regulatedlacZ fusion. However, the Fur-CI857 fusion was unable to complement the growth defect of anE. coli fur mutant on fumarate and succinate. The C-terminal domain of Fur, when fused to the N-terminus of CI857, repressed a P, -regulatedlacZ fusion, indicating dimerization of the chimeric protein, which is a prerequisite for Cl activity. Both fusion proteins were fully active under both iron-rich and iron-poor growth conditions. We conclude that the N-terminal domain of Fur is involved in recognition of the Fur-responsive promoter and the C-terminus mediates oligomerization of the repressor.     

Development of yeast strains for the efficient utilisation of starch: evaluation of constructs that express α-amylase and glucoamylase separately or as bifunctional fusion proteins

Eight constructions involving the Bacillus subtilis -amylase gene (amyE), a mouse pancreatic -amylase cDNA (AMY2) and an Aspergillus awamori glucoamylase cDNA (glaA) were prepared: three fusion genes, involving one -amylase and the glucoamylase, two double-cassette plasmids (expressing one or other -amylase and the glucoamylase) and three single-cassette plasmids, expressing the individual coding sequences. Following transformation of each plasmid into Saccharomyces cerevisiae, a plate test revealed that the largest starch hydrolysis halo was produced by the strain bearing the B. subtilis -amylase/glucoamylase fusion (BsAAase/GAase), and the smallest halo by the one expressing the mouse pancreatic -amylase/glucoamylase fusion (MAAase/GAase). When assayed for enzymatic activity in liquid medium, the strains bearing the fusion and the double-cassette plasmids involving B. subtilis -amylase and the glucoamylase exhibited both enzymic activities. Moreover, the BsAAase/GAase hybrid was able to adsorb and digest raw starch. The MAAse/GAase fusion protein was found to exhibit only -amylase activity. Finally, the capacity to grow on soluble and corn starch was tested in liquid medium for the strains bearing plasmids coding for the fusion proteins and the separate enzymes. The strain carrying the double-cassette BsAAase + GAase, which produced one of the smallest hydrolysis haloes in the place test, showed the best performance, not only in digesting soluble and corn starch but also in using all of the hydrolysis products for growth. The transformant bearing the BsAAase/GAase fusion was able to grow on soluble starch, but not on corn starch.     

The b Subunit of Escherichia coli ATP Synthase

The b subunit of ATP synthase is a major component of the second stalk connecting the F₁and F₀ sectors of the enzyme and is essential for normal assembly and function. The156-residue b subunit of the Escherichia coli ATP synthase has been investigated extensivelythrough mutagenesis, deletion analysis, and biophysical characterization. The two copies ofb exist as a highly extended, helical dimer extending from the membrane to near the top ofF₁, where they interact with the subunit. The sequence has been divided into four domains:the N-terminal membrane-spanning domain, the tether domain, the dimerization domain, andthe C-terminal -binding domain. The dimerization domain, contained within residues 60–122,has many properties of a coiled-coil, while the -binding domain is more globular. Sites ofcrosslinking between b and the a, , , and subunits of ATP synthase have been identified,and the functional significance of these interactions is under investigation. The b dimer mayserve as an elastic element during rotational catalysis in the enzyme, but also directly influencesthe catalytic sites, suggesting a more active role in coupling.     

Alpha-factor leader sequence-directed transport of Escherichia coli beta-galactosidase in the secretory pathway of Saccharomyces cerevisiae

Summary The constuction of two fused genes is described. One involves the in-frame fusion of the yeast prepro--factor coding sequence, and the Escherichia coli lac Z gene. The second gene fusion utilizes a 103 bp yeast invertase NH₂-terminal coding sequence at the fusion junction of the hybrid gene described above. The gene fusions, under the control of the -factor promoter, expressed active -galactosidase in haploid yeast cells. The activity could be regulated in a temperature-sensitive sir3 mutant. The incorporation of the invertase coding sequence at the MF1-lacZ fusion junction provided significantly higher levels of -galactosidase activity. A substantial quantity of the hybrid proteins generated from the gene fusions was primarily localized in the intracellular membranes of yeast cells, while a processed form could be secreted into the periplasm.A portion of this work appeared in Biotechnology Progress (Das and Shultz 1986) as proceedings of the symposium on Industrial Scale Protein Purification, held at the annual meeting of the Institute of Chemical Engineers in Miami Beach, Fla, USA on November 4, 1986     

Chromosome banding in amphibia

A cytogenetic study performed on a population of the South American leptodactylid frog Eleutherodactylus maussi revealed multiple sex chromosomes of the X₁X₁X₂X₂/X₁X₂Y (=XXAA/XXA^Y) type. The diploid chromosome number is 2n=36 in all females and 2n=35 in most males. The multiple sex chromosomes originated by a centric fusion between the original Y chromosome and a large autosome. In male meiosis the X₁X₂Y (=XXA^Y) multiple sex chromosomes form a classical trivalent configuration. E. maussi is the first species discovered in the class Amphibia that is distinguished by a system of multiple sex chromosomes. Only one single male was found in the population with 2n=36 chromosomes and lacking the Y-autosomal fusion. This karyotype (XYAA) is interpreted as the ancestral condition, preceding the occurrence of the Y-autosome fusion.by H.C. Macgregor     

Expression of an active spinach acyl carrier protein-I/protein-A gene fusion

A synthetic gene encoding spinach acyl carrier protein I (ACP-I) was fused to a gene encoding the Fc-binding portion of staphylococcal protein A. This gene fusion, under the control of the P_R promoter, was expressed at high levels in Escherichia coli producing a 42 kDa fusion protein. This fusion protein was phosphopantethenylated in E. coli. In vitro the ACP portion of the fusion protein was able to participate in acyl ACP synthetase reactions, plant malonyl-CoA:ACP transacylase (MCT) reactions, and plant fatty acid synthetase (FAS) reactions. Inhibitory effects of high ACP concentrations on in vitro plant FAS were observed with the unfused ACP-1 but not with the fusion protein. As with unfused ACP-I, the fusion protein was a poor substrate for E. coli FAS reactions. When injected into rabbits, the fusion protein was also able to generate antiserum to spinach ACP-I.     

“Erussica”, the intergeneric fertile somatic hybrid developed through protoplast fusion between Eruca sativa Lam. and Brassica juncea (L.) Czern.

Summary Hypocotyl calli-derived protoplasts of two cultivars of Brassica juncea (2n=36), a major oil-seed crop, were fused with normal as well as -irradiated mesophyll protoplasts of Eruca sativa (2n=22). The irradiation of the Eruca fusion partner increased the plating efficiency as well as the morphogenic potentiality of the fusion products over the normal fusion. Fertile plants could be regenerated from such fusion products. Analysis of 63 out of 181 plants regenerated showed that, indeed, 11 somatic hybrids (2n=58) and 10 partial somatic hybrids (chromosome number ranged between 50 and 56) had been obtained. Pollen viability (0%–82.9%) and seed set (0%–50%) of the hybrids indicated them to be useful for future studies.     

A vector for the site-directed,genomic integration of foreign DNA into soybean root-nodule bacteria

A non-essential DNA region carrying two different repeated sequences (RS3 and RS9) adjacent to a nitrogen fixation (nif) gene cluster has been identified previously in Bradyrhizobium japonicum strain 110. In closely related B. japonicum strains a similar genomic arrangement was found. We constructed a mobilizable plasmid vector carrying RS3 and RS9, and a kanamycin resistance cassette (nptII gene) plus suitable cloning sites inserted between the two repeated sequences. Using this vector (pRJ1035), stable integration of a lacZ gene fusion into the B. japonicum genomic RS region was achieved. The resulting strain yielded more than 10-fold higher -galactosidase activity in soybean root nodules as compared to a B. japonicum strain carrying the same lacZ fusion on a pRK290-based plasmid.     

The genetic defect in the various types of human β-galactosidase deficiency

Summary Gene localization studies revealed the presence of two structural -galactosidase (GAL) loci on the human chromosomes 3 and 22 (de Wit et al., 1979). To determine the function of these genes, proliferating hybrid cell lines were isolated following fusion of fibroblasts from two different patients with a GAL deficiency and Chinese hamster cells. The hybrids were analyzed electrophoretically and immunologically.Fibroblasts from a patient with an adult type of GAL deficiency associated with a neuraminidase deficiency were used for the first fusion. No evidence for a structural GAL mutation was found in these hybrids. The absence of a structural GAL mutation is consistent with a primary defect in neuraminidase in this adult patient.Fibroblasts from a patient with the infantile type 1 G_M1-gangliosidosis were used for the second fusion. It is concluded that the human determinants present in the isolated hybrid lines occur in heteropolymeric man-Chinese hamster molecules. The heteropolymeric isoenzyme in (+3–22) hybrids is very labile and is sensitive to neuraminidase treatment. Therefore it is concluded that the infantile type 1 patient is mutated in the structural GAL gene on chromosome 3. Because this patient has a primary defect in G_M1-GAL, the GAL gene on chromosome 3 is apparently a G _M1-GAL gene. Interaction of the two GAL loci results in an additional band of GAL activity on electrophoresis. This suggests that the gene on chromosome 22 is also a structural G _M1-GAL gene.     

Somatic hybrid plants obtained by protoplast fusion between Citrus sinensis and Poncirus trifoliata

Summary Somatic hybrid plants of Rutaceae were obtained by protoplast fusion between Citrus sinensis Osb. (Trovita orange) and Poncirus trifoliata. Protoplasts isolated from embryogenic cells of C. sinensis and from leaves of P. trifoliata, and the culture of fusion products in the presence of high concentrations of sucrose were essential requirements for the selection of hybrids. Green globular embryoids derived from protoplasts resulted in the regeneration of trifoliate plants. Other morphological characters of these plants were intermediate between both parents. The chromosome number in one of the hybrid plants was 36, which was the sum of C. sinensis (2n=18) and P. trifoliata (2n=18). EcoRI restriction analysis of rDNA confirmed the presence of parental nuclear DNAs in the hybrid.     

Autosomal fragmentations and fusions in odonata and their evolutionary implications

The fragmentation of autosomes in the karyotypes ofEnallagma cyathigerum (Charp.) (n=1415),Mecistogaster sp. (n=15),Hetaerina rosea Sel. (n=14),Libellula depressa L. (n=1213),Orthetrum coerulescens (Fabr.) (n=1213),Diplacodes bipunctata (Br.) (n=1315) andD. haematodes (Burm.) (n=1213) is discussed. Original material ofMecistogaster andHetaerina was not available.Fragmentations were found to be the only way in which the recombination index is obligatorily changed (increased) in dragonflies. In at least five out of the seven cases considered the chromosome number is not stabilised: cells in which fragmentation occurred and those in which it did not are found in the same individual (Enallagma, Libellula, Orthetrum, Diplacodes). Fragmentation results in an increase of chromosome number (a) up to the family type number level (Libellula, Orthetrum, D. haematodes), (b) above the latter (Hetaerina) and (c) above any chromosome number ever reported in dragonflies (Mecistogaster, D. bipunctata). In our material the element or elements formed by fragmentation have always the size and usually also the heterochromatic features of the m-chromosomes, irrespective of the presence or absence of the latter in the original complement.A review of species in which fusions of autosomes were recorded or can be assumed is given.Orthetrum brachiale (Beauv.) (n=11) is added to the list. The situation in this species and inSympetrum eroticum Sel. is discussed in detail.The most essential differences between the fusion of (an) autosome (s) with the sex element on one hand (cf.Kiauta, 1969), and the fusion of two or more autosomes on the other, lies in the observations: (a) that in the former case, fused and unfused complements occur in one individual, whereas the latter is specifically characteristic and occurs in all cells, all individuals and all populations of the species, and (b) that autosomal fusion results in an increase of chiasma frequency, due to which the recombination index in secondarily low-n complements remains the same as it was in the primary high-n sets, or becomes even higher (O. brachiale, S. eroticum).Autosomal fragmentation (found so far in some advanced forms only) is considered as a character of phylogenetic advancement. Autosomal fusion, on the other hand, does not have any relation with phylogeny.     

Genetic mapping by means of protoplast fusion in Bacillus subtilis

Summary A new mapping method involving protoplast fusion in Bacillus subtilis is described. Protoplasts from an isogenic standard marker strain containing purA and from a strain containing both purB and the marker, x, to be mapped were fused with polyethylene glycol, and purA ⁺ purB ⁺ fusants were selected. After isolation of single colonies and determination of unselected markers, marker x was mapped between two standard markers. This method was fully applicable to PBS1-resistant strains (e.g., lyt strains). The results obtained by protoplast fusion, conventional transformation and/or lysed protoplast transformation indicated that a lyt strain, Ni15, contained two new autolysin-minus mutations (lyt-151 and lyt-152). The properties of lyt-15 are also discussed.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - SMM 0.5 M sucrose, 0.02 M MgCl₂, 0.02 M maleate buffer, pH 6.5     

Asymmetric somatic hybridization between tomato (Lycopersicon esculentum Mill) and gamma-irradiated potato (Solanum tuberosum L.): a quantitative analysis

We analyzed 110 asymmetric fusion products, obtained by fusion of hygromycin-resistant tomato protoplasts and gamma-irradiated kanamycin-resistant potato protoplasts that expressed -glucuronidase (GUS). The fusion products were selected for resistance to both antibiotics, and were subsequently analyzed for their shoot regeneration potential, GUS activity, expression of two potato isoenzymes, chloroplast type, total genomic DNA content, and relative genomic composition. No viable plants could be obtained and the calli were highly polypoid. All hybrids expressed GUS activity, whereas they displayed a large variation with respect to the other traits.     

Relationship between the grain pattern in the wood,domain pattern and pattern of growth activity in the storeyed cambium of trees

Summary The relationship between the arrangement of cell events occurring in cambium in a definite configuration and the grain pattern of wood was investigated. Taking into consideration the growth activity of fusiform cell ends, a model of a migrating morphogenetic wave determining an event configuration was made. Waves of length =1 m for the periods T=2 years and T=3 years and waves of lengths =l m and =0.04 m for the period T=10 years were considered. On the model, events from successive annual rings, conventionally comprising 10 cell layers each, were summed. In this way, event maps were obtained. For wave =4 mm, the domain pattern on the modelled map was compatible with the grain pattern. The domain pattern for the wave =1 m was impossible to recreate because the wave migrated too fast. In this case, the pattern of event configuration, incompatible with the grain pattern, formed microareas, which were not domains.     

Protoplast fusion between Lactobacillus casei and Lactobacillus acidophilus

Summary From the fusion between Lactobacillus casei and Lactobacillus acidophilus, 8 fusants were selected: Four were able to ferment maltose, lactose, galactose and mannose, but two had greater abilities of acid production than parents. Increased values of up to 7.6–8 % in -galactosidase activity were obtained from two when compared to that of L. acidophilus, whereas another 2 had activities of 800 and 548 nmol/mg protein/min comparable to that of L casei giving a value of 400 nmol/mg protein/min in phospho--galactosidase activity.     

Expression of the RAD1 and RAD3 genes of Saccharomyces cerevisiae is not affected by DNA damage or during the cell division cycle

Summary The RAD1 and RAD3 genes of Saccharomyces cerevisiae are required for excision repair of UV damaged DNA. In addition, the RAD3 gene is essential since rad3 deletions are recessive lethals. We have examined the induction of the RAD1 and RAD3 genes by DNA damage and during the cell division cycle. We have made fusions of the RAD1 and RAD3 genes with the Escherichia coli lacZ gene encoding -galactosidase. -galactosidase activity was measured in a Rad⁺ yeast strain containing the RAD1-lacZ or the RAD3-lacZ fusion, either in a multicopy replicating plasmid or as a single copy integrant resulting from transformation with an integrating plasmid which transforms yeast by homologous recombination in the yeast genome. No induction of -galactosidase activity occurred after ultraviolet light (UV) or 4-nitroquinoline-1-oxide (NQO) treatment. Haploid cells of mating type a were synchronized by treatment with factor and -galactosidase activity was determined during different cell cycle stages. No change in -galactosidase activity was observed in the strain containing the RAD1-lacZ or the RAD3-lacZ fusion integrated in the yeast genome.     

In-vitro processing of yeast α-factor leader fusion proteins using a soluble yscF (Kex2) variant

Summary The Saccharomyces cerevisiae KEX2 gene encodes the membrane-bound endoprotease yscF, which is responsible for the site-specific endoproteolytic cleavages at pairs of basic amino acid residues in the -factor precursor. In order to obtain soluble yscF activity, a mutant KEX2 gene lacking 600 bp coding for the C-terminal 200 amino acids was constructed. Expression of the truncated KEX2 gene in yeast led to the secretion of an active soluble yscF protein (yscF_s). The soluble yscF protein is able to efficiently cleave heterologous protein precursors in-vitro, as demonstrated for -factor leader-hIGF1 and -factor leader-hirudin fusion proteins. Offprint requests to: P. G. Seeboth     

Mutation to rifampicin resistance at the beginning of the RNA polymerase beta subunit gene in Escherichia coli

Summary The unusual recombinant plasmid pRC19 carrying the N-terminal fragment of the Escherichia coli RNA polymerase rpoB gene was found to specify high level rifampicin resistance of E. coli cells. Sequence analysis of this plasmid revealed one substitution only: transversion GT, leading to amino acid substitution Val¹⁴⁶Phe. This mutational change marks the second domain of the subunit involved in rifampicin binding.