Development of a versatile procedure based on natural transformation for marker-free targeted genetic modification in Streptococcus thermophilus

A versatile natural transformation protocol was established for and successfully applied to 18 of the 19 Streptococcus thermophilus strains tested. The efficiency of the protocol enables the use of in vitro-amplified mutagenesis fragments to perform deletion or insertion of large genetic fragments. Depending on the phenotype linked to the mutation, markerless mutants can be selected either in two steps, i.e., resistance marker insertion and excision using an adapted Cre-loxP system, or in one step using a powerful positive screening procedure as illustrated here for histidine prototrophy.     

<Emphasis Type="Italic">α</Emphasis>-Amylase inhibitor-1 gene from <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> expressed in <Emphasis Type="Italic">Coffea arabica</Emphasis>plants inhibits α-amylases from the coffee berry borer pest

Background  

Coffee is an important crop and is crucial to the economy of many developing countries, generating around US70 billion per year. There are 115 species in the < i > Coffea < /i > genus, but only two, < i > C. arabica < /i > and < i > C. canephora < /i > , are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer ( < i > Hypotheneumus hampei < /i > ), is responsible for worldwide annual losses of around US70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an α-amylase inhibitor gene (α-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants.     

Genomic comparison of P-type ATPase ion pumps in Arabidopsis and rice

Members of the P-type ATPase ion pump superfamily are found in all three branches of life. Forty-six P-type ATPase genes were identified in Arabidopsis, the largest number yet identified in any organism. The recent completion of two draft sequences of the rice (Oryza sativa) genome allows for comparison of the full complement of P-type ATPases in two different plant species. Here, we identify a similar number (43) in rice, despite the rice genome being more than three times the size of Arabidopsis. The similarly large families suggest that both dicots and monocots have evolved with a large preexisting repertoire of P-type ATPases. Both Arabidopsis and rice have representative members in all five major subfamilies of P-type ATPases: heavy-metal ATPases (P1B), Ca2+-ATPases (endoplasmic reticulum-type Ca2+-ATPase and autoinhibited Ca2+-ATPase, P2A and P2B), H+-ATPases (autoinhibited H+-ATPase, P3A), putative aminophospholipid ATPases (ALA, P4), and a branch with unknown specificity (P5). The close pairing of similar isoforms in rice and Arabidopsis suggests potential orthologous relationships for all 43 rice P-type ATPases. A phylogenetic comparison of protein sequences and intron positions indicates that the common angiosperm ancestor had at least 23 P-type ATPases. Although little is known about unique and common features of related pumps, clear differences between some members of the calcium pumps indicate that evolutionarily conserved clusters may distinguish pumps with either different subcellular locations or biochemical functions.     

Metastasis of bronchogenic carcinoma to the thumb

Bone metastasis in the hand is rare. The etiology is quite different from that of metastasis to other bones; bronchogenic carcinoma is by far the most frequent case. Distal phalanges are mainly involved with irregular osteolysis and cortical destruction. Differential diagnosis of phalangeal metastasis includes osteomyelitis, rheumatoid arthritis and gout. The prognosis is always that of metastatic bronchial cancer with an average survival of three months. Treatment may involve distal digital amputation or antalgic radiotherapy. A case of bronchogenic carcinoma with metastasis to the thumb is presented. The metastasis was located in the distal phalanx of the left thumb. The primary tumor was located in the lung. Treatment consisted of amputation. The overall survival was five months.     

A Plant Plasma Membrane ATP Binding Cassette–Type Transporter Is Involved in Antifungal Terpenoid Secretion

ATP binding cassette (ABC) transporters, which are found in all species, are known mainly for their ability to confer drug resistance. To date, most of the ABC transporters characterized in plants have been localized in the vacuolar membrane and are considered to be involved in the intracellular sequestration of cytotoxins. Working on the assumption that certain ABC transporters might be involved in defense metabolite secretion and their expression might be regulated by the concentration of these metabolites, we treated a Nicotiana plumbaginifolia cell culture with sclareolide, a close analog of sclareol, an antifungal diterpene produced at the leaf surface of Nicotiana spp; this resulted in the appearance of a 160-kD plasma membrane protein, which was partially sequenced. The corresponding cDNA (NpABC1) was cloned and shown to encode an ABC transporter. In vitro and in situ immunodetection showed NpABC1 to be localized in the plasma membrane. Under normal conditions, expression was found in the leaf epidermis. In cell culture and in leaf tissues, NpABC1 expression was strongly enhanced by sclareolide and sclareol. In parallel with NpABC1 induction, cells acquired the ability to excrete a labeled synthetic sclareolide derivative. These data suggest that NpABC1 is involved in the secretion of a secondary metabolite that plays a role in plant defense.     

Microsatellite variation in Drosophila melanogaster and Drosophila simulans: a reciprocal test of the ascertainment bias hypothesis

Interspecific comparisons of microsatellite loci have repeatedly shown that the loci are longer and more variable in the species from which they are derived (the focal species) than are homologous loci in other (nonfocal) species. There is debate as to whether this is due to directional evolution or to an ascertainment bias during the cloning and locus selection processes. This study tests these hypotheses by performing a reciprocal study. Eighteen perfect dinucleotide microsatellite loci identified from a Drosophila simulans library screen and 18 previously identified in an identical Drosophila melanogaster library screen were used to survey natural populations of each species. No difference between focal and nonfocal species was observed for mean PCR fragment length. However, heterozygosity and number of alleles were significantly higher in the focal species than in the nonfocal species. The most common allele in the Zimbabwe population of both species was sequenced for 31 of the 36 loci. The length of the longest stretch of perfect repeat units is, on average, longer in the focal species than in the non-focal species. There is a positive correlation between the length of the longest stretch of perfect repeats and heterozygosity. The difference in heterozygosity can thus be explained by a reduction in the length of the longest stretch of perfect repeats in the nonfocal species. Furthermore, flanking-sequence length difference was noted between the two species at 58% of the loci sequenced. These data do not support the predictions of the directional-evolution hypothesis; however, consistent with the ascertainment bias hypothesis, the lower variability in nonfocal species is an artifact of the microsatellite cloning and isolation process. Our results also suggest that the magnitude of ascertainment bias for repeat unit length is a function of the microsatellite size distribution in the genomes of different species.      

Prospects for estimating nucleotide divergence with RAPDs

The technique of random amplification of polymorphic DNA (RAPD), which is simply polymerase chain reaction (PCR) amplification of genomic DNA by a single short oligonucleotide primer, produces complex patterns of anonymous polymorphic DNA fragments. The information provided by these banding patterns has proved to be of great utility for mapping and for verification of identity of bacterial strains. Here we consider whether the degree of similarity of the banding patterns can be used to estimate nucleotide diversity and nucleotide divergence. With haploid data, fragments generated by RAPD-PCR can be treated in a fashion very similar to that for restriction-fragment data. Amplification of diploid samples, on the other hand, requires consideration of the fact that presence of a band is dominant to absence of the band. After describing a method for estimating nucleotide divergence on the basis of diploid samples, we summarize the restrictions and criteria that must be met when RAPD data are used for estimating population genetic parameters.