Cotransformation of Aspergillus nidulans: a tool for replacing fungal genes

Summary When a non-selected DNA sequence was added during the transformation of amdS320 deletion strains of Aspergillus nidulans with a vector containing the wild-type amdS gene the AmdS⁺ transformants were cotransformed at a high frequency. Cotransformation of an amdS320, trpC801 double mutant strain showed that both the molar ratio of the two vectors and the concentration of the cotransforming vector affected the cotransformation frequency. The maximum frequency obtained was defined by the gene chosen as selection marker for transformation. Cotransformation was used to induce a gene replacement in A. nidulans. An amdS320 strain was transformed to AmdS⁺ and cotransformed with a DNA fragment containing a fusion between a non-functional A. nidulans trpC gene and the Escherichia coli lacZ gene. Ten AmdS⁺, LacZ⁺ transformants with a Trp^– mutant phenotype were selected. All of these strains could be transformed with a functional copy of the A. nidulans trpC gene, but only two strains yielded TrpC⁺ transformants which, with a low frequency, had a LacZ^– phenotype. These latter transformants had also lost the AmdS⁺ phenotype. Southern blotting analysis of DNA from these transformants confirmed the inactivation of the wild-type trpC gene, but revealed that amdS vector sequences were also involved in the gene replacement events.     

Genetic and molecular mapping of the pma1 mutation conferring vanadate resistance to the plasma membrane ATPase from Saccharomyces cerevisiae

Summary In the yeast Saccharomyces cerevisiae, the pma1 mutations confers vanadate-resistance to H⁺-ATPase activity when measured in isolated plasma membranes. In vivo, the growth of pma1 mutants is resistant to Dio-9, ethidium bromide and guanidine derivatives. This phenotype was used to man the pma1 mutation adjacent to LEU1 gene on chromosome VII. From a cosmid library of a wild-type Saccharomyces cerevisiae genome, a large 30 kb DNA fragment was isolated by complementation of a leu1-pma1 double mutant. A 5 kb HindIII fragment was subcloned and it restored both Leu⁺ and Pma⁺ phenotypes after integrative transformation. The restriction map of the 5 kb HindIII fragment and Southern blot analysis reveal that the cloned fragment contains the entire structural gene for the plasma membrane ATPase and the 5 end of the adjacent LEU1 gene. The pma1 mutation conferring vanadate-resistance is thus located in the structural gene for the plasma membrane ATPase.Publication n^o 2456 from the Biology Directorate of the Commission of European Communities     

Localization by in situ hybridization of a low copy chimaeric resistance gene introduced into plants by direct gene transfer

Summary An in situ hybridization method was developed for detecting single or low copy number genes in metaphase chromosomes of plants. Using as a probe ³H-labelled plasmid pABDI, which confers kanamycin resistance (Km^r) to transformed cells. DNA introduced into the plant genome by direct gene transfer was detected with a high efficiency: about 60% to 80% of interphase and metaphase plates showed a strong signal. The insertion site of the Km^r gene in two independent transformants was localised on different homologous chromosome pairs. This result independently confirmed previous genetic data which had indicated that transformed DNA was integrated into plant chromosomes in single blocks.     

Rapid immunofluorescent determination of cells in the S phase in pea root meristems: An alternative to autoradiography

Photosystem II (PS II) activity and the localization of ribulose-l,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) were studied in primary leaves of young maize plants ( Zea mays L. cv. Fronica) by tetra-nitro-blue-tetrazoliumchloride reduction and immunolocalization, respectively. In tissue of 3-day-old plants all chloroplasts were structurally identical. From day 4 they developed into their typical appearance of mesophyll and bundle sheath chloroplasts. First PS II-activity was present in both types of chloroplasts. From day 4 it disappeared in bundle sheath chloroplasts concomitant with the loss of grana. RuBP carboxylase on the other hand was only present in bundle sheath chloroplasts at all stages of development. Thus, the control of the development of the photosystems and the Calvin cycle enzymes seem to differ.     

Genetic diversity within and among 11 juvenile populations of green alder (Alnus crispa) in Canada

Germinated seeds from 11 populations of green alder [ Alnus crispa (Ait.) Pursh] sampled in four Canadian provinces were analysed for electrophoretically demonstrable diversity of 10 enzymes encoded by 15 structural loci. Of these, nine were polymorphic, and on average, 52% of the loci per population were polymorphic. Assuming a diploid model of expression, average level of expected heterozygosity was 0.11 with nearly all populations in Hardy-Weinberg equilibrium for the set of polymorphic loci analysed. No significant inbreeding and associated subpopulation structuring were noted. Rates of gene flow appeared high within and among populations. Although little divergence was observed among populations, genetic and geographical distances between populations were related. Discriminant and cluster analyses revealed a pattern of genetic variation associated with geography. Populations from northern Quebec were poorly differentiated, whereas western populations from Alberta exhibited a larger degree of genetic differentiation. Introgresive hybridization with the sympatric species Alnus sinuata (Regel) Rydberg and partial isolation in the West are suggested as an explanation for this larger differentiation. The occurrence and significance of rare alleles is discussed in relation to the importance of geographical distance in the process of population differentiation in this species.     

Internode length in Pisum. Action of gene lw

Jolly, C. J., Reid, J. B. and Ross, J. J. 1987. Internode length in Pisum. Action of gene lw.
Mutant K29 of Pisum sativum L. is shown to possess a recessive gene at a new locus, lw , which results in reduced internode length, delayed flowering and increased symptoms of water congestion compared with the parental cv. Torsdag. The interaction of gene lw with the internode length genes na, le, la and cry ⁵ is examined. Extracts from the shoots of Iw plants are shown to contain similar levels of gibberellin (GA)-like substances to comparable Lw plants, but Iw plants do not elongate to the same extent as Lw plants when treated with GA₁₉ GA₁₉, or GA₂₀. The effect of gene Iw is not graft-transmissible. Unlike essentially isogenic dwarf lines possessing the GA-synthesis genes le, Ih or Is, lw plants show a relative increase in elongation similar to Torsdag in response to photoperiod extensions from sources rich in far-red light. These results suggest that gene lw probably does not reduce elongation by influencing GA-synthesis and that the response to photoperiod extensions with far-red light may depend on the level of GA.     

Inheritance of resistance to potato y viruses in Phaseolus vulgaris L

Summary Resistance to watermelon mosaic virus-2 in Phaseolus vulgaris L. is conferred by two distinct dominant alleles at independent loci. Based on segregation data one locus is designated Wmv, the other, Hsw. The dominant allele Wmv from cv. Great Northern 1140 prevents systemic spread of the virus but viral replication occurs in inoculated tissue. In contrast, Hsw confers both local and systemic resistance to WMV-2 below 30C. At higher temperatures, plants that carry this allele in the absence of modifying or epistatic factors develop systemic veinal necrosis upon inoculation with the virus that results in rapid death. Patho-type specificity has not been demonstrated for either allele; both factors confer resistance to every isolate tested. A temperature-sensitive shift in epistasis is apparent between dominant alleles at these loci. Because Hsw is very tightly linked if not identical to the following genes for hypersensitivity to potyviruses I, (bean common mosaic virus), Bcm, (blackeye cowpea mosaic virus), Cam, (cowpea aphid-borne mosaic virus) and Hss (soybean mosaic virus), parental, reciprocal dihybrid F₁ populations, and selected F₃ families were inoculated with each of these viruses and held at 35 C. F₁ populations developed vascular necrosis completely or primarily limited to inoculated tissue, while F₃ families from WMV-2-susceptible segregates were uniformly susceptible to these viruses. The relationship between Hsw, Wmv and other genes for potyvirus resistance suggest patterns in the evolution of resistance and viral pathogenicity. Characterization of the resistance spectrum associated with each factor provides an additional criterion to distinguish genes for plant virus resistance.     

Nucleic acids in mummified plant seeds: screening of twelve specimens by gel-electrophoresis,molecular hybridization and DNA cloning

Summary Twelve seed specimens of varying ages and from different archaeological sites were analyzed for the presence of polymerized DNA and RNA. Amongst the samples tested, one of Vitis vinifera from an archaeological site in Iran (2,000–3,000 B.C.) was found to be completely devoid of nucleic acids. Zea mais seeds of Precolumbial age from Peru (about 800 A.D.) contained depolymerized DNA and RNA. Samples of Vitis vinifera and Rubus sp. from a Lombard archaeological site (800 A.D.) as well as radiocarbon dated seeds from the site of the Spring Sanctuary near Metaponto (I–IV century B.C.) were found to contain polymerized DNA and rRNA bands. However the electrophoretic properties of the rRNAs in one case and hybridization experiments performed with cloned seed DNA in the other, clearly demonstrated that the polymerized nucleic acids were not of plant origin.     

Structural organization of cholera toxin gene and its expression in an environmental non-pathogenic strain ofVibrio cholerae

Non-pathogenic, environmental strain ofVibrio cholerae, ELTOR Ogawa EW6 carries a copy of the cholera toxin gene in its chromosome. Restriction enzyme digestion followed by Southern blot analysis revealed that the structure of the cholera toxin gene in this organism is different from that found in the virulent strains. The xbaI site which has been found to be conserved in the cholera toxin of the virulent strains examined so far, is absent here. Results of the RNA dot blot analysis indicated that the cholera toxin gene in EW6 is transcribed much less efficiently compared to the cholera toxin gene present in the virulent strainVibrio cholerae classical Inaba 569B.     

Endosymbiotic origin and codon bias of the nuclear gene for chloroplast glyceraldehyde-3-phosphate dehydrogenase from maize

Summary The nuclei of plant cells harbor genes for two types of glyceraldehyde-3-phosphate dehydrogenases (GAPDH) displaying a sequence divergence corresponding to the prokaryote/eukaryote separation. This strongly supports the endosymbiotic theory of chloroplast evolution and in particular the gene transfer hypothesis suggesting that the gene for the chloroplast enzyme, initially located in the genome of the endosymbiotic chloroplast progenitor, was transferred during the course of evolution into the nuclear genome of the endosymbiotic host. Codon usage in the gene for chloroplast GAPDH of maize is radically different from that employed by present-day chloroplasts and from that of the cytosolic (glycolytic) enzyme from the same cell. This reveals the presence of subcellular selective pressures which appear to be involved in the optimization of gene expression in the economically important graminaceous monocots.