Loss of activity in the secreted form of Escherichia coli haemolysin caused by an rfaP lesion in core lipopolysaccharide assembly

A transposon mutant of Escherichia coli 5K was isolated which reduced 10- to 50-fold the secreted extracellular haemolytic activity of cells carrying the complete hlyCABD operon while leaving unaffected the intracellular haemolytic activity and the levels of intracellular and extracellular haemolysin protein, HlyA. The transposon insertion was identified within the rfaP gene (required for attachment of phosphate-containing substituents to the lipopolysaccharide inner core), and extracellular haemolytic activity was restored in trans by the intact rfaP gene. The toss in cytolytic activity of the secreted HlyA protein was not related to the HlyC-directed acylation of the protoxin. Activity of the secreted toxin was restored by chaotropic agents and during rate-zonal centrifugation the mutant-secreted HlyA migrated as a larger species than the wild type. The results indicate that the rfaP mutation affects the aggregation behaviour of the active toxin during or following the signal peptide-independent secretion process.     

Sex determination in plants

Sex determination is an important developmental event in the life cycle of all sexually reproducing plants. Recent studies of sex determination in many plant species, from ferns to maize, have been fruitful in identifying the diversity of genetic and epigenetic factors that are involved in determining the sex of the flower or individual. In those species amenable to genetic analysis, significant progress has been made toward identifying mutations that affect sex expression. By studying the interactions among these genes, pictures of how sex-determining signals are perceived to activate or repress male- or female-specific genes are emerging.     

Cloning of the chromosomal determinants encoding hemolysin production and mannose-resistant hemagglutination in Escherichia coli.

We have cloned the chromosomal hemolysin determinants from Escherichia coli strains belonging to the four O-serotypes O4, O6, O18, and O75. The hemolysin-producing clones were isolated from gene banks of these strains which were constructed by inserting partial Sau3A fragments of chromosomal DNA into the cosmid pJC74. The hemolytic cosmid clones were relatively stable. The inserts were further subcloned either as SalI fragments in pACYC184 or as BamHI-SalI fragments in a recombinant plasmid (pANN202) containing cistron C (hlyC) of the plasmid-encoded hemolysin determinant. Detailed restriction maps of each of these determinants were constructed, and it was found that, despite sharing overall homology, the determinants exhibited minor specific differences in their structure. These appeared to be restricted to cistron A (hlyA), which is the structural gene for hemolysin. In the gene banks of two of these hemolytic strains, we could also identify clones which carried the genetic determinants for the mannose-resistant hemagglutination antigens Vb and VIc. Both of these fimbrial antigens were expressed in the E. coli K-12 clones to an extent similar to that observed in the wild-type strains. These recombinant cosmids were rather unstable, and, in the absence of selection, segregated at a high frequency.     

An extra species of lysine transfer ribonucleic acid in polyoma virus-transformed cells in tissue culture

Isoaccepting tRNAs from various mouse cells were fractionated on columns of benzoylated DEAE cellulose. Lysine tRNA from mouse embryo, adult mouse liver and kidney, primary mouse embryo cells in tissue culture, and an established tissue culture line of mouse fibroblasts (3T3) has two peaks of isoaccepting tRNA; lysine tRNA from two established lines of polyoma virus-transformed cells contains an additional peak of lysine tRNA. The extra peak in transformed cells comprises about 25% of the acceptor capacity for lysine. It is stable to denaturation and renaturation and can be chromatographed, stripped of lysine, recharged, and rechromatographed. The extra peak is present in tRNA from transformed cells and absent in tRNA from normal cells regardless of whether the lysyl-tRNA ligase used for aminoacylation is from normal or transformed cells. Isoaccepting tRNAs for arginine, leucine, serine, valine, histidine, and tyrosine reveal similar profiles for the various tRNAs from normal and transformed cells.     

In vitro cultivation and electron microscopy characterization of Trachipleistophora anthropophthera isolated from the cornea of an AIDS patient

We describe an in vitro culture technique for a microsporidian isolated from the corneal biopsy of an HIV-infected patient. The corneal biopsy was inoculated into a monolayer culture of fibroblasts derived from newborn mouse brain and incubated at 37 degrees C in an atmosphere of 5% CO2. Minimum essential medium supplemented with 2% fetal bovine serum appeared to be an optimum medium for growth and maintenance of the parasite and for production of large numbers of spores. This microsporidian was identified as Trachipleistophora anthropophthera based on ultrastructural features. It forms two types of sporophorous vesicles and two types of spores simultaneously: polysporous vesicle type I with eight or more oval spores, 3.7-4.0 microm by 2.0-2.3 microm, and bisporous vesicle type II with two round spores, 1.7-2.2 microm by 1.6-2.0 microm in size.     

The phiX174 protein J mediates DNA packaging and viral attachment to host cells

Packaging of viral genomes into their respective capsids requires partial neutralization of the highly negatively charged RNA or DNA. Many viruses, including the Microviridae bacteriophages phiX174, G4, and alpha3, have solved this problem by coding for a highly positively charged nucleic acid-binding protein that is packaged along with the genome. The phiX174 DNA-binding protein, J, is 13 amino acid residues longer than the alpha3 and G4 J proteins by virtue of an additional nucleic acid-binding domain at the amino terminus. Chimeric phiX174 particles containing the smaller DNA-binding protein cannot be generated due to procapsid instability during DNA packaging. However, chimeric alpha3 and G4 phages, containing the phiX174 DNA-binding protein in place of the endogenous J protein, assemble and are infectious, but are less dense than the respective wild-type species. In addition, host cell attachment and native gel migration assays indicate surface variations of these viruses that are controlled by the nature of the J protein. The structure of alpha3 packaged with phiX174 J protein was determined to 3.5A resolution and compared with the previously determined structures of phiX174 and alpha3. The structures of the capsid and spike proteins in the chimeric particle remain unchanged within experimental error when compared to the wild-type alpha3 virion proteins. The amino-terminal region of the phiX174 J protein, which is missing from wild-type alpha3 virions, is mostly disordered in the alpha3 chimera. The differences observed between solution properties of wild-type phiX174, wild-type alpha3, and alpha3 chimera, including their ability to attach to host cells, correlates with the degree of order in the amino-terminal domain of the J protein. When ordered, this domain binds to the interior of the viral capsid and, thus, might control the flexibility of the capsid. In addition, the properties of the phiX174 J protein in the chimera and the results of mutational analyses suggest that an evolutionary correlation may exist between the size of the J protein and the stoichiometry of the DNA pilot protein H, required in the initial stages of infection. Hence, the function of the J protein is to facilitate DNA packaging, as well as to mediate surface properties such as cell attachment and infection.     

An auxin transport independent pathway is involved in phosphate stress-induced root architectural alterations in Arabidopsis. Identification of BIG as a mediator of auxin in pericycle cell activation

Arabidopsis (Arabidopsis thaliana) plants display a number of root developmental responses to low phosphate availability, including primary root growth inhibition, greater formation of lateral roots, and increased root hair elongation. To gain insight into the regulatory mechanisms by which phosphorus (P) availability alters postembryonic root development, we performed a mutant screen to identify genetic determinants involved in the response to P deprivation. Three low phosphate-resistant root lines (lpr1-1 to lpr1-3) were isolated because of their reduced lateral root formation in low P conditions. Genetic and molecular analyses revealed that all lpr1 mutants were allelic to BIG, which is required for normal auxin transport in Arabidopsis. Detailed characterization of lateral root primordia (LRP) development in wild-type and lpr1 mutants revealed that BIG is required for pericycle cell activation to form LRP in both high (1 mm) and low (1 microm) P conditions, but not for the low P-induced alterations in primary root growth, lateral root emergence, and root hair elongation. Exogenously supplied auxin restored normal lateral root formation in lpr1 mutants in the two P treatments. Treatment of wild-type Arabidopsis seedlings with brefeldin A, a fungal metabolite that blocks auxin transport, phenocopies the root developmental alterations observed in lpr1 mutants in both high and low P conditions, suggesting that BIG participates in vesicular targeting of auxin transporters. Taken together, our results show that auxin transport and BIG function have fundamental roles in pericycle cell activation to form LRP and promote root hair elongation. The mechanism that activates root system architectural alterations in response to P deprivation, however, seems to be independent of auxin transport and BIG.     

Genetic transformation and regeneration of mature tissues of woody fruit plants bypassing the juvenile stage

Regeneration and transformation systems from mature plant material of woody fruit species have to be achieved as a necessary requirement for the introduction of useful genes into specific cultivars and the rapid evaluation of resulting horticultural traits. We report here, for the first time, a procedure for genetic transformation and regeneration of mature tissues of woody plants that overcomes the long juvenile periods and high heterozygosity that are characteristic of most of these species. An improved regeneration frequency from mature explants was obtained by invigoration of the plant material through grafting of mature buds on juvenile seedlings. Co-cultivation of the explants in feederplates after inoculation with Agrobacterium tumefaciens resulted in enhanced transformation frequencies. Furthermore, in vitro shoot-tip grafting of the regenerated mature shoots on seedling rootstocks provided a rapid and efficient system for plant production. Citrus is the most extensivel y grown fruit crop worldwide and sweet orange (Citrus sinensis L. Osbeck) accounts for approximately 70% of the Citrus total production. Mature transgenic sweet orange plants have been obtained, which flowered and bore fruit in 14 months     

A new peirosaurid (Crocodilyformes) from the Late Cretaceous (Turonian–Coniacian) of Patagonia,Argentina

Peirosauridae is composed of mid- to large-sized terrestrial mesoeucrododylian crocodyliforms distributed throughout Gondwanan landmasses. Here we describe a new peirosaurid that comes from the upper levels of the Portezuelo Formation (Turonian–Coniacian, Upper Cretaceous) from Loma de la Lata, Neuquén Province, Argentina. This specimen consists of some associated bones belonging to a single individual. In order to facilitate comparisons, we recognise two different peirosaurid morphotypes based on skull shape: broad- and narrow-snouted taxa. The new taxon may be related to broad-snouted taxa, especially Gasparinisuchus peirosauroides. The new taxon here reported has strong heterodont dentition when compared with other peirosaurids. As in related forms, the fourth dentary tooth is caniniform, very large, acute and transversely compressed (much more than other peirosaurids), and the anterior dentary teeth have less globular, sharp serrated crowns. Large interalveolar spaces are present between both mandibular and maxillary teeth, a trait only observed on the new taxon. With this addition, we elevate the number of Patagonian peirosaurids to four. Moreover, it represents together with Lomasuchus palpebrosus the second peirosaurid species described for the Portezuelo Formation.