Giuseppe Damiani

Flavobacteria as intracellular symbionts in cockroaches

Animal cells are the sole habitat for a variety of bacteria. Molecular sequence data have been used to position a number of these intracellular microorganisms in the overall scheme of eubacterial evolution. Most of them have been classified as proteobacteria or chlamydiae. Here we present molecular evidence placing an intracellular symbiont among the flavobacteria-bacteroides. This microorganism inhabits specialized cells in the cockroach fat body and has been described as a mutualistic endosymbiont of uncertain phylogenetic position. The small subunit ribosomal DNA of these bacteria was analysed after polymerase chain reaction amplification to investigate their phylogeny. The endosymbionts of five species of cockroaches were found to make up a coherent group with no close relatives within the eubacterial phylum defined by the flavobacteria. In addition, the relationships among the endosymbionts, as revealed by DNA sequence data, appeared to be congruent with the host taxonomic relationships. Based on the host fossil record, a tentative calibration of the nucleotide substitution rate for the cockroach flavobacteria gave results congruent with those obtained for the aphid endosymbiotic proteobacteria.

The establishment of intracellular symbiosis in an ancestor of cockroaches and termites

All cockroaches examined so far have been found to harbour a bacterial endosymbiont in specialized cells of the fat body, whereas Mastotermes darwiniensis is the only termite currently known to intracellular symbiont. The localization and mode of transmission of these bacteria are surprisingly similar, but so far no data have been published on their phylogenetic relationships. To address this issue, molecular sequence data were obtained from the genes encoding the small subunit ribosomal RNA of the M. darwiniensis endosymbiont, and compared with those obtained from endosymbionts of seven species of cockroaches. Molecular phylogenetic analysis unambiguously placed all these bacteria among the flavobacteria-bacteroides, indicating that the endosymbiont of M. darwiniensis is the sister group to the cockroach endosymbionts examined. Additionally, nucleotide divergence between the endosymbionts appears to be congruent with the palaeontological data on the hosts’s evolution. These results support previous claims that the original infection occurred in an ancestor common to cockroaches and termites. A loss of endosymbionts should subsequently have occurred in all termite lineages, except that which gave rise to M. darwiniensis.

Use of random amplified polymorphic DNA (RAPD) for generating specific DNA probes for microorganisms

We report the rapid generation of DNA probes for several Azospirillum strains. This method does not require any knowledge of the genetics and/or the molecular biology of the organism (genome) to be investigated. The procedure is based on the generation of random amplified polymorphic DNA (RAPD) fingerprints using primers with an embedded restriction site. The amplification product(s) peculiar to one strain or common to two or more strains can be purified, cloned, sequenced and used as molecular probes in hybridization experiments for the detection and identification of microorganisms. We have tested this methodology in the nitrogen‐fixing bacterium Azospirillum by amplyfing the total DNA extracted from several Azospirillum strains. We have used amplification bands with different specificity as molecular probes in hybridization experiments performed on amplified DNA. Results obtained have demonstrated the usefulness of this methodology for Azospirillum. Its use in microbial ecology studies as a general strategy to generate specific DNA probes is also discussed.

Cloning of histidine genes of Azospirillum brasilense: Organization of the ABFH gene cluster and nucleotide sequence of the hisB gene

SummaryA cluster of four Azospirillum brasilense histidine biosynthetic genes, hisA, hisB, hisF and hisH, was identified on a 4.5 kb DNA fragment and its organization studied by complementation analysis of Escherichia coli mutations and nucleotide sequence. The nucleotide sequence of a 1.3 kb fragment that complemented the E. coli hisB mutation was determined and an ORF of 624 nucleotides which can code for a protein of 207 amino acids was identified. A significant base sequence homology with the carboxyterminal moiety of the E. coli hisB gene (0.53) and the Saccharomyces cerevisiae HIS3 gene (0.44), coding for an imidazole glycerolphosphate dehydratase activity was found. The amino acid sequence and composition, the hydropathic profile and the predicted secondary structures of the yeast, E. coli and A. brasilense proteins were compared. The significance of the data presented is discussed.

Evidence for genomic changes in transgenic rice (Oryza sativa L.) recovered from protoplasts.

The occurrence of genomic modifications in transgenic rice plants recovered from protoplasts and their transmission to the self-pollination progeny has been verfied with the random amplified polymorphic DNA (RAPD) approach. The plant was the Indica-type rice (Oryza sativa L.) cultivar Chinsurah Boro II. The analysed material was: (1) microspore-derived embryogenic rice cells grown in suspension culture, (2) transgenic plants recovered from protoplasts produced from the cultured cells and (3) the self-pollination progeny (two successive generations) of the transgenic plants. DNA purified from samples of these materials was PCR-amplified with different random oligonucleotide primers and the amplification products were analysed by agarose gel electrophoresis. Band polymorphism was scored and used in band-sharing analyses to produce a similarity matrix. Relationships among the analysed genomes were expressed in a dendrogram.The extensive DNA changes evidenced in cultured cells demonstrate the occurrence of somaclonal variation in the material used to produce protoplasts for gene transfer. Quantitatively reduced DNA changes were also found in the resulting transgenic plants and i their self-pollination progenies.While confirming the stability of the foreign gene in transgenic plants, this work gives molecular evidence for the occurrence of stable genomic changes in transgenic plants and points toin vitro cell culture as the causative agent. RAPDs are shown to be a convenient tool to detect and estimate the phenomenon at the molecular level. The methodology is also proposed as a fast tool to select those transgenic individuals that retain the most balanced genomic structure and to control the result of back-crosses planned to restore the original genome.

Ligation overcomes terminal underrepresentation in multiple displacement amplification of linear DNA

The amount of DNA available for informative genomic studies is often limiting. Thus, several methods have been developed to achieve a whole genome amplification (WGA). Particu-larly promising is a variant, called multiple displacement amplification (MDA) (1,2), which exploits the high processivity of Φ29 phage DNA polymerase and suffers an amplifi-cation bias significantly lower than previous, PCR-based WGA (3).Even though MDA-DNA has been successfully used for many applica-tions (1,4–7), some problems have been reported. For example, sequences near the ends of linear chromosomes appear underrepresented after MDA (6,8,9). Both defective priming events and abortive chain terminations could contribute to this problem, which limits the applications of MDA and jeopardizes its use on short linear DNA (e.g., cDNA and degraded genomes from forensic, archeological, and fetal origin) (10). In these cases, the terminal underrepresentation would cause the loss of substantial information.We studied this problem using λ phage DNA (48.5 kb; Promega Biosciences, San Luis Obispo, CA, USA). MDA was performed using the Genomiphi

Comparison of an improved RAPD fingerprinting with different typing methods for discriminating clinical isolates of Staphylococcus spp.

Different epidemiological markers were used to characterize 2 Staphylococcus epidermidis and 8 Staphylococcus aureus strains isolated from patients with severe infections. We compared random amplified polymorphic DNA (RAPD) fingerprints, biotypes, antibiotic assays, plasmid profiles and chromosomal DNA restriction endonuclease analysis (REA). Data analysis based on numerical taxonomy methods indicates that RAPD and REA give similar results allowing a good discrimination of the two species and of each isolate. The RAPD method is easier and faster than REA, but the reproducibility of RAPD fingerprints obtained in independent experiments can be problematic. We have found simple technical devices to improve the reproducibility of the RAPD procedure which is therefore a very useful tool in epidemiology for identification and characterization of Staphylococcus spp.

Phylogenetic Studies of the Genus Azospirillum

The 16S rDNA of seventeen Azospirillum strains, fourteen of which assigned to one of known A. amazonense, A. brasilense, A. halopraeferens, A. irakense and A. lipoferum species, and other three of uncertain taxonomic collocation, was sequenced and analysed after polymerase chain reaction amplification, in order to investigate the phylogenetic relationships at the intra-generic and super-generic level. Moreover the use of histidine biosynthetic genes and of anonymous sequences generated by RAPD technique to infer phylogenetic relationships among Azospirillum strains is also described. The relationships among the five species, as revealed by DNA sequence data, appeared to be congruent with other molecular and phenotypic results, showing that the genus Azospirillum constitutes a phylogenetically separate entity in which A. brasilense and A. lipoferum are very close species, whereas the species A. amazonense is the most divergent.

Evolutionary Meaning, Functions and Morphogenesis of Branching Structures in Biology

A causal explanation of the regularity and invariance of biological forms must relate to morphogenetic generative laws, to allometric constraints of growth processes, to biunique relations of an organism with environment and other organisms, to the pattern of evolutionary processes, and to physical causes as the forces present in the environment and the mechanical properties of the available building materials. The evaluation of the relative importance of these different causes is not always easy. Are evolutionary trends of organic forms the results of functional adaptation or of random processes? A general model to explain the ontogenetic and phylogenetic upward trend of complexity of biological branching structures is proposed. The function of these structures in living organisms is to distribute or to gather biological material and physical entities. Optimization processes produced by natural selection led to minimization of the networks length (the Steiner problem) and of the amount of information needed for the construction of these structures. What is the optimization method chosen by nature? Experimental data and computer simulations suggest a simple way to generate minimized network: sensitive entities spread or concentrate in a surface or in a volume responding to morphogenetic gradients of physical forces or of chemical substances. Recursive local rules of repulsion or attraction leads to the formation of fractal, branching, vascular and global networks. Slight changes of morphogenetic fields and of responsiveness of sensitive entities influence shapes of produced patterns. Natural selection allows survival of the biological structures more suitable for their physiological functions: to distribute or to gather something in an economic (cost functions minimization), uniform (space-filling) and size-independent (self-similar scaling) way. Therefore biological branching structures are the results of functional adaptation processes. Computer simulations by means of genetic algorithms based on the same optimization method chosen by nature may be applied successfully to solve a wide variety of scientific and engineering problems.

Evaluation of gene expression in human lymphocytes activated in the presence of melatonin

The effect of melatonin on the expression of genes previously correlated to T lymphocyte activation (HLA-DRB, thymosin beta 10 (beta-Tim)) and to Lymphokine Activated Killer (LAK) activity (beta-Tim, Tumour Rejection Antigen (TRA 1), nRap 2) was investigated in phytohemagglutinin (PHA)-stimulated human lymphocyte cultures. The aim was to find an enhancing effect of this substance on anti-tumoral immune defences as suggested by studies on tumour progression in mice and clinical immunotherapy trials in humans. mRNA obtained from melatonin-treated and -untreated PHA-stimulated lymphocytes was retrotranscribed and amplified by RT-PCR using primers based on the sequences of the selected genes. The results suggest that melatonin does not increase T and LAK cell responses: in fact, a reduction in the transcription of all the considered genes was observed. These data are correlated with the antiproliferative effect of melatonin observed in in vitro treated lymphocytes.