Genome-wide identification and organization of seed storage protein genes of <Emphasis Type="Italic">Cannabis sativa</Emphasis>

Hemp (Cannabis sativa L.) seeds have been recognized as a nutritional protein source for humans and animals. In this study, gene families encoding precursor polypeptides of three storage protein classes, including six 11S edestin, two 2S albumin and one 7S vicilin-like genes were identified and characterized from an inbred line of hemp. All edestins showed typical 11S globulin features but based on the amino acid composition, they were grouped in three edestin types (type1, -2 and -3). Genes encoding edestin type1 and -3 were very close to each other in a DNA fragment of 16 071 bp, whereas the two isoforms of edestin type2 were linked on a different DNA fragment of 8 232 bp and arranged in a tailto- tail fashion. All edestin types were very rich in arginine and glutamic acid, but edestin type3 was the richest in cysteine and methionine. Regarding the 2S albumin (Cs2S) two genes were identified in a fragment of 13 738 bp in a tail-to-head array. Finally, only one 7S-vicilin like gene (Cs7S) that exhibited typical 7S vicilin features, such as the presence of two cupin domains and several N-glycosylation sites, was isolated. Southern blot hybridization is in agreement with the number of genes isolated, and real-time qPCR analysis revealed that all genes are expressed in the seed. The highest expression was observed for edestin type1 (CsEde1) and Cs2S, whereas the lowest expression was detected for Cs7S. The results of this study provide a complete overview of the genes encoding hemp storage proteins and significantly advance our knowledge on the organization of these gene families.     

Distribution of highly repeated DNA sequences in species of the genus Lens Miller.

Multiple-target fluorescence in situ hybridization (FISH) was applied on mitotic chromosomes of seven Lens taxa using two highly repetitive sequences (pLc30 and pLc7) isolated from the cultivated lentil and the multigene families for the 18S-5.8S-25S (pTa71) and 5S rRNA (pTa794) from wheat simultaneously as probes. The number and location of pLc30 and pLc7 sites on chromosomes varied markedly among the species, whereas the hybridization pattern of 5S rDNA and 18S-5.8S-25S rDNA was less variable. In general, each species showed a typical FISH karyotype and few differences were observed among accessions belonging to the same species, except for the accessions of Lens odemensis. The most similar FISH karyotype to the cultivated lentil is that of Lens culinaris subsp. orientalis, whereas Lens nigricans and Lens tomentosus are the two species that showed the most divergent FISH patterns compared with all taxa for number and location of pLc30 and 18S-5.8S-25S rDNA sites.     

Isolation and characterisation of an lpa (low phytic acid) mutant in common bean (Phaseolus vulgaris L.)

Phytic acid is considered as one of the major antinutritional compounds in cereal and legume seeds. The development of lpa (low phytic acid) grains, resulting in increased mineral cation availability, is considered a major goal in the improvement of the nutritional quality of seed crops, especially those largely consumed in developing countries. From a mutagenised population of common bean we isolated a homozygous lpa mutant line (lpa-280-10) showing, compared to wild type, a 90% reduction of phytic acid, a 25% reduction of raffinosaccharides and a much higher amount of free or weakly bound iron cations in the seed. Genetic analysis showed that the lpa character is due to a recessive mutation that segregates in a monogenic, Mendelian fashion. Germination tests performed using varying ageing or stress conditions, clearly showed that the bean line lpa-280-10 has a better germination response than the wild type. These data, together with those obtained from 2 years of agronomic trials showing that the mutant seed yield is close to that of its parents and other evidence, indicate that the new lpa-280-10 mutation might be the first devoid of visible macroscopic negative effects in plants, pods and seeds. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chlamydia trachomatis serovar distribution and other sexually transmitted coinfections in subjects attending an STD outpatients clinic in Italy

We studied the prevalence of Chlamydia trachomatis (CT) urogenital infection and the distribution of different genotypes in a non-selected STD population of 1625 patients, evaluating presence of coinfections with other sexually transmitted diseases. Each patient was bled to perform serological tests for syphilis and HIV, then urethral or endocervical swabs were obtained for the detection of CT and Neisseria gonorrhoeae by culture. DNA extracted from remnant positive swabs was amplified by omp1 Nested PCR and products were sequenced. Total prevalence of CT infection was 6.3% (103/1625), with strong differences between men and women (11.4% vs 3.9%, P<0.01). Clinical symptoms and coinfections were much more frequent in men than in women (P<0.01). The most common serovar was E (prevalence of 38.8%), followed by G (23.3%), F (13.5%) D/Da (11.6%) and J (4.8%). Serovars distribution was statistically different between men and women (P=0.042) and among patients with or without coinfection (P=0.035); patients infected by serovar D/Da showed the highest coinfection rate. This study can be considered a contribution in increasing knowledge on CT serovar distribution in Italy. Further studies are needed to better define molecular epidemiology of CT infection and to investigate its correlation with other STDs.     

A family of dispersed repeats in the genome of Vicia faba: structure, chromosomal organization, redundancy modulation, and evolution

A family of repeated DNA sequences of about 1200 bp in length and bordered by well-conserved, 18 bp inverted repeats (VfB family) was found in the nuclear genome of Vicia faba. The structure, chromosomal organization, redundancy modulation and evolution of these sequences were investigated. They are enriched in A+T base pairs (about 40% G+C) and lack any obvious internally repeated motif. A 64%–73% nucleotide sequence identity was found when pairwise comparisons between VfB sequences were carried out (average 69%). Direct repeats were not found to flank the inverted repeats that border these DNA sequences. The results obtained by hybridizing VfB repeats to Southern blots of V. faba genomic DNA digested with EcoRI indicated that these DNA elements are interspersed in the genome. The appearance of bands in these Southern blots and comparison of the structure of the sequences that flank different VfB elements showed that these repeats might be part of other, longer repeated DNA sequences. A high degree of dispersion throughout the genome was confirmed by cytological hybridization, which showed VfB sequences to be scattered along the length of all chromosomes and to be absent or rare only at heterochromatic chromosomal regions. These sequences contribute to intraspecific alterations of genomic size. Indeed, dot-blot hybridizations proved that their redundancy, which is positively correlated with the overall amount of nuclear DNA in each accession, varies between V. faba land races (27×10³–230×10³ copies per 1C DNA). Southern blot hybridization of VfB repeats to restriction endonuclease-digested genomic DNAs of V. faba, V. narbonensis, V. sativa, Phaseolus coccineus, Populus deltoides, and Triticum durum revealed nucleotide sequence homology of these DNA elements, whatever the stringency conditions, only to the DNAs of Vicia species, and to a reduced extent to the DNAs of V. narbonensis and V. sativa compared with that of V. faba. It is concluded that VfB repeats might be descended from mobile DNA elements and contribute to change genomic size and organization during evolution. Received: 10 September 1998; in revised form: 12 May 1999 / Accepted: 19 May 1999     

Genomic organization and phylogenetic relationships in the genus Dasypyrum analysed by Southern and in situ hybridization of total genomic and cloned DNA probes

Molecular cytogenetic methods have been used to study the controversial phylogenetic relationships between the species Dasypyrum villosum (L.) Candargy (2n=2x=14) and D. breviaristatum (Lindb. f.) Frederiksen (2n=4x=28). Using total genomic DNA from the two species as probes for in situ hybridization to chromosomes, we found that the pericentromeric regions of the chromosome arms of both species are similar, while distal regions show substantial differences. Two dispersed repetitive DNA sequences were isolated: pDbKB45 is distributed along the chromosomes but amplified in the subtelomeric regions of D. breviaristatum chromosomes, while pDbKB49, in both species, is less amplified in terminal regions. Size-separated restriction enzyme digests of DNA showed many repetitive fragments, but few in common between the two species. After probing Southern transfers with D. breviaristatum genomic DNA, all lanes showed similar hybridization patterns although one extra small band was evident in the D. breviaristatum lanes. In contrast, probing with D. villosum DNA showed very substantial differences between the two species. Genomic in situ hybridization to meiotic metaphases from an interspecific hybrid showed seven bivalents of D. breviaristatum origin and seven univalents from D. villosum. We also analysed the physical organization of 5S rDNA, 18S-25S rDNA and a tandemly repeated sequence from rye. Our data support an autotetraploid origin for D. breviaristatum, but its genome and that of D. villosum show extensive differences, so the tetraploid is unlikely to be directly derived from D. villosum. Received: 29 March 1996; in revised form: 28 December 1996 / Accepted: 2 February 1997     

A MiRNA Signature for Defining Aggressive Phenotype and Prognosis in Gliomas

Gliomas represent a disparate group of tumours for which there are to date no cure. Thus, there is a recognized need for new diagnostic and therapeutic approaches based on increased understanding of their molecular nature. We performed the comparison of the microRNA (miRNA) profile of 8 WHO grade II gliomas and 24 higher grade tumours (2 WHO grade III and 22 glioblastomas) by using the Affymetrix GeneChip miRNA Array v. 1.0. A relative quantification method (RT-qPCR) with standard curve was used to confirm the 22 miRNA signature resulted by array analysis. The prognostic performances of the confirmed miRNAs were estimated on the Tumor Cancer Genome Atlas (TCGA) datasets. We identified 22 miRNAs distinguishing grade II gliomas from higher grade tumours. RT-qPCR confirmed the differential expression in the two patients'' groups for 13 out of the 22 miRNAs. The analysis of the Glioblastoma Multiforme (GBM) and Lower Grade Glioma (LGG) datasets from TCGA demonstrated the association with prognosis for 6 of those miRNAs. Moreover, in the GBM dataset miR-21 and miR-210 were predictors of worse prognosis in both univariable and multivariable Cox regression analyses (HR 1.19, p = 0.04, and HR 1.18, p = 0.029 respectively). Our results support a direct contribution of miRNAs to glioma cancerogenesis and suggest that miR-21 and miR-210 may play a role in the aggressive clinical behaviour of glioblastomas.     

Evolutionary analysis of the APA genes in the <Emphasis Type="Italic">Phaseolus</Emphasis> genus: wild and cultivated bean species as sources of lectin-related resistance factors?

The APA (Arcelin/Phytohemagglutinin/α-Amylase inhibitor) gene family is composed of various members, present in Phaseolus species and coding for lectin and lectin-related seed proteins having the double role of storage and defense proteins. Here members of the APA family have been identified by immunological, functional, and molecular analyses and representative genes were sequenced in nine wild species of Phaseolus. All taxa possessed at least one member of the true lectin gene. No arcelin type sequences have been isolated from the species examined. Among the wild species studied, only P. costaricensis contained an α-amylase inhibitor (α-AI). In addition P. augusti, P. maculatus, P. microcarpus, and P. oligospermus showed the presence of the lectin-related α-amylase inhibitor-like (AIL) genes and α-AI activity. Data from Southern blot analysis indicated the presence of only one lectin gene in P. parvulus and P. filiformis, while an extensive gene duplication of the APA locus was found in the other Phaseolus species. Phylogenetic analysis carried out on the nucleotide sequences showed the existence of two main clusters and clearly indicated that lectin-related genes originated from a paralogous duplication event preceding the development of the ancestor to the Phaseolus genus. The finding of detectable α-AI activity in species containing AIL genes suggests that exploiting APA genes variability in the Phaseolus genus may represent a valuable tool to find new members that may have acquired insecticidal activities. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.