T-cell receptor variable alpha (TCRAV) polymorphisms in European, Chinese, South American, AfroCaribbean, and Gambian populations

 Interactions involving the T-cell receptor (TCR) and major histocompatibility complex (MHC) are fundamental to the generation of a specific immune response. The study of interpopulation differences in TCR genes may identify those genes which are subject to selection, and also provides useful information for future genetic studies in these populations. In this study we present analysis of five TCRAV polymorphisms, for V5S1, V6S1, V8S1, V17S1, and V21S1 loci in five human populations by single-strand conformational polymorphism (SSCP) analysis. Caucasian, Chinese, Gambian, AfroCaribbean, and South American Indians (Mapuches) showed marked interpopulation variation for both the silent (V5S1, V17S1, and V21S1) and coding (V6S1 and V8S1) polymorphisms. In general the alleles were conserved in the different populations, but new, additional variants were found for V5S1 and V17S1 in Gambians and Caucasians. V6S1 overall showed the highest nucleotide diversity, and V6S1 genotype distributions were skewed away from expected values in Chinese and Mapuches. Analysis of allelic associations showed a general lack of linkage disequilibrium between the loci, which was reflected by the absence of strong population-specific haplotypes. Received: 22 April 1997 / Revised: 9 July 1997     

Genetic characterization of clinical and agri-food isolates of multi drug resistant <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Heidelberg from Canada

Background  

Salmonella enterica serovar Heidelberg ranks amongst the most prevalent causes of human salmonellosis in Canada and an increase in resistance to extended spectrum cephalosporins (ESC) has been observed by the Canadian Integrated Program for Antimicrobial Resistance Surveillance. This study examined the genetic relationship between S. Heidelberg isolates from livestock, abattoir, retail meat, and clinical human specimens to determine whether there was a link between the emergence of MDR S. Heidelberg in chicken agri-food sources and the simultaneous increase of MDR S. Heidelberg in human clinical samples.     

Staphylococcus aureus virulence attenuation and immune clearance mediated by a phage lysin‐derived protein

New anti‐infective approaches are much needed to control multi‐drug‐resistant (MDR) pathogens, such as methicillin‐resistant Staphylococcus aureus (MRSA). Here, we found for the first time that a recombinant protein derived from the cell wall binding domain (CBD) of the bacteriophage lysin PlyV12, designated as V12CBD, could attenuate S. aureus virulence and enhance host immune defenses via multiple manners. After binding with V12CBD, S. aureus became less invasive to epithelial cells and more susceptible to macrophage killing. The expressions of multiple important virulence genes of S. aureus were reduced 2.4‐ to 23.4‐fold as response to V12CBD. More significantly, V12CBD could activate macrophages through NF‐κB pathway and enhance phagocytosis against S. aureus. As a result, good protections of the mice from MRSA infections were achieved in therapeutic and prophylactic models. These unique functions of V12CBD would render it a novel alternative molecule to control MDRS. aureus infections.     

Organoarsenical tolerance in Sphingobacterium wenxiniae,a bacterium isolated from activated sludge

Organoarsenicals enter the environment from biogenic and anthropogenic sources. Trivalent inorganic arsenite (As(III)) is microbially methylated to more toxic methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)) that oxidize in air to MAs(V) and DMAs(V). Sources include the herbicide monosodium methylarsenate (MSMA or MAs(V)), which is microbially reduced to MAs(III), and the aromatic arsenical roxarsone (3-nitro-4-hydroxybenzenearsonic acid or Rox), an antimicrobial growth promoter for poultry and swine. Here we show that Sphingobacterium wenxiniae LQY-18^T, isolated from activated sludge, is resistant to trivalent MAs(III) and Rox(III). Sphingobacterium wenxiniae detoxifies MAs(III) and Rox(III) by oxidation to MAs(V) and Rox(V). Sphingobacterium wenxiniae has a novel chromosomal gene, termed arsU1. Expressed in Escherichia coli arsU1 confers resistance to MAs(III) and Rox(III) but not As(III) or pentavalent organoarsenicals. Purified ArsU1 catalyses oxidation of trivalent methylarsenite and roxarsone. ArsU1 has six conserved cysteine residues. The DNA sequence for the three C-terminal cysteines was deleted, and the other three were mutated to serines. Only C45S and C122S lost activity, suggesting that Cys45 and Cys122 play a role in ArsU1 function. ArsU1 requires neither FMN nor FAD for activity. These results demonstrate that ArsU1 is a novel MAs(III) oxidase that contributes to S. wenxiniae tolerance to organoarsenicals.     

Phylogenetic study of sponge associated bacteria from the Lakshadweep archipelago and the antimicrobial activities of their secondary metabolites

Marine ecosystem of the Lakshadweep archipelago is unique and known to have a very high degree of biodiversity with a number of endemic flora and fauna. The present study focuses to isolate the endosymbiotic microorganism from sponges and its effectiveness against marine ornamental fish pathogens. The sponges were collected from Agatti island of Lakshadweep archipelago and identified as Clathria procera, Sigmadocia fibulata and Dysidea granulosa. In which, 15 different types of bacteria were isolated and screened against marine ornamental fish pathogens (A. hydrophila, Vibrio alginolyticus, V. harveyii, V. parahaemolyticus and Pseudomonas fluorescens). The strain S25 was found as potential bacteria based on their antimicrobial activity against the fish pathogens. Molecular identification of the potential strain (S25) of the 16S rRNA gene showed 99% identity with Acinetobacter sp. The sequenced 16 s rRNA gene with 1,081 bp in length was submitted in NCBI Genbank and Accession was obtained (GenBank Accession number HM004071). The strain exhibited high similarity (99%) with the 16S rRNA gene of Acinetobacter calcoaceticus from GenBank database. Crude extract obtained with acetone and ethyl acetate from extracellular products of S25 showed significant antimicrobial activity by disc diffusion assay using 1,500 μg/ml of crude extract. Extracellular metobolite of A. calcoaceticus was extracted by shake flask method and the crude extract was partially purified by thin layer chromatography. Partially purified crude extract showed significant inhibition zone of antimicrobial activity (A. hydrophila, V. alginolyticus, V. parahaemolyticus) and less similar activity against V. harveyii and P. fluorescens. This is the first report on A. calcoaceticus isolated from sponges of Lakshadweep archipelago and the studies are underway to characterize and purify the antimicrobial compounds of the potential bacteria.     

Arsenate phytoextraction abilities of one-year-old tree species and its effects on the nutritional element content in plant organs

Abstract

The aim of the study was to evaluate the As phytoextraction potential of four tree species: Acer pseudoplatanus L., Betula pendula Roth., Quercus robur L., and Ulmus laevis Pall. in light of their prospective use in the phytoremediation of arsenate [As(V)] contaminated soils. The content of nutritional elements: B, Ca, K, Mg, Na, Si, P, and S was also analyzed. The trees were grown for 1?month in hydroponic cultures (Knop medium) supplemented with As(V), (1?mM). The results showed that the highest As accumulation efficiency was characterized by B. pendula (BCF = 0.87) and Q. robur (BCF = 0.5). Betula pendula accumulated about 80% of As in its roots (TF = 0.22) whereas Q. robur accumulated more than 60% of As in its shoots (TF = 1.60). The other tree species accumulated significantly lower amounts of As, more than 60% of which collected in their shoots. As(V) phytoextraction led to a significantly lower level of P and S in the roots of all tested tree species. Betula pendula seems promising for phytostabilisation and Q. robur for phytoextraction of As(V) from contaminated soils. The obtained results confirm the accumulation and translocation of As(V), as well as the acquisition of nutritional elements by the selected tree species.     

Asymbiotic germination of ornamental <Emphasis Type="Italic">Vanda:</Emphasis> in vitro germination and development of three hybrids

The majority of potted flowering orchids sold in the United States are Phalaenopsis hybrids. However, demand for other genera will undoubtedly increase as consumers become accustomed to growing orchids in the home. The objective of this study was to compare the germination and development of three Vanda hybrids, a genus with mass market potential. Specifically, we evaluated whether hybrids with different pedigrees have significantly different culture requirements. After 12 weeks culture, differences in germination and development were found among the three hybrids screened. Generally, seeds of Vanda Motes Primrose × Ascocenda Tavivat (hybrid S014) showed higher germination (82.0–95.3%) than seeds of V. Paki × (V. tessellata × V. cristata; hybrid S005) or (V. Joan Warne × V. Paki) × V. Loke (hybrid S013) when cultured on Knudson C, half-strength Murashige & Skoog, or PhytoTechnology Orchid Seed Sowing Medium under three photoperiods (8/16 h, 12/12 h, 16/12 h; light/dark). Protocorms of S005 and S013 did not develop beyond Stage 3 (first leaf present) under any of the conditions tested, while S014 seeds cultured on PhytoTechnology Orchid Seed Sowing Medium developed to Stage 4 (one leaf and ≥1 roots present; 0.5–0.6%) and 5 (two leaves and ≥1 roots present; 2.7–5.6%) under all photoperiods tested. All hybrids appeared to undergo similar early development (Stages 0–3). Limited advanced development of Hybrids S005 and S013 may be due either to inadequate culture conditions or low vigor of these hybrids. These data indicate that commercial production of some Vanda hybrids may be limited by slow growth and development. Efforts to breed hybrids which germinate and develop rapidly may be required to improve the mass market potential of Vanda hybrids.     

Newly characterized genetic polymorphism of uropepsinogen group A (PGA) using both isoelectric focusing and immunoblotting

Summary Genetic polymorphism of uropepsinogen group A (PGA) was characterized in human urine using a technique involving both polyacrylamide gel isoelectric focusing and immunoblotting with an anti-PGA antibody. PGA was clearly separable into five fractions, termed I to V in order of decreasing anodal mobility. The most slowly migrating fraction V was composed of F (fast) and/or S (slow) band(s). The population frequencies of the three patterns of fraction V (F, FS, and S) and family studies indicated that PGA V is controlled by a pair of alleles, PGA V ^* F and PGA V ^* S, at a single autosomal locus, and that both are codominant. The frequencies of the genes are 0.07 for PGA V ^* F and 0.93 for PGA V ^* S.     

Conventional and molecular methods to detect bacterial pathogens in mussels

Aim: To detect Aeromonas spp., Salmonella spp., Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus in mussels and water samples from a farming area, conventional and molecular methods were applied to enrichment cultures. Methods and Results: The aerolysin gene (aero) of Aeromonas spp., the invasion plasmid antigen B (ipaB) gene of Salmonella spp., the enterotoxin secretion protein (epsM) gene of V. cholerae, the species‐specific region of 16S rRNA gene of V. vulnificus, the 16S–23S rDNA (IGS) gene of V. parahaemolyticus and the pR72H fragment of V. parahaemolyticus were amplified by multiplex polymerase chain reaction (PCR) assays on DNA extracted from enrichment cultures. The haemolysin gene (tdh) of pathogenic V. parahaemolyticus was also amplified. Conventional culture method allowed the isolation of V. parahaemolyticus and V. vulnificus from water and mussels. The genes aero, epsM and 16S rRNA of V. vulnificus were occasionally detected in the enrichment cultures. In mussels, the ipaB and IGS genes were detected from June to September and from April to November, respectively. All genes, except aero, were amplified from mussels collected in September, when pathogenic V. parahaemolyticus (tdh+) strains were also isolated. Conclusions: Multiplex‐PCR assays were more sensitive and faster than conventional procedures. Significance and Impact of the Study: The results emphasize the need of an accurate and rapid detection of bacterial pathogens in mussels to protect human health.     

A potential bacterial biocontrol agent,strain S2V2 against pathogenic marine <Emphasis Type="Italic">Vibrio</Emphasis> in aquaculture

We isolated a marine bacterium strain S2V2 which inhibited the growth of pathogenic marine Vibrio spp. The aims of this research were to identify a new antibiotic-producing marine bacterium strain S2V2, and evaluate its spectrum activity and pathogenic property. Analysis of 16S rDNA sequence placed strain S2V2 in the genus Pseudoalteromonas, but the sequence similarity was low (95.46%) implying the strain might be a new species in this genus. Strain S2V2 inhibited the growth of 67.9% of 28 Vibrio strains tested. This strain inhibited V. alginolyticus, V. anguillarum, V. fluvialis, V. harveyi, V. metschnikovii, V. splendidus, V. ordalii, V. parahaemolyticus, and V. vulnificus, but inactive against V. campbellii, Aeromonas hydrophyla and Staphylococcus aureus. Strain S2V2 produced extracellular non proteinaceous antibacterial substances. The highest antibacterial activity was found when strain S2V2 was cultured for 96 h in ZoBell broth medium. An artificial infection to post larvae of Lithopenaeus vanname indicated that strain S2V2 was a non pathogenic bacterium. Non pathogenic property and specific antibacterial activity against a broad range of fish pathogenic marine Vibrio of strain S2V2 suggest that this strain is a prospective source of unique antibiotic and a potential biocontrol agent in marine aquaculture.     

Rapid screening of <Emphasis Type="Italic">Salmonella enterica</Emphasis>serovars Enteritidis,Hadar, Heidelberg and Typhimurium using a serologically-correlative allelotyping PCR targeting the O and H antigen alleles

Background  

Classical Salmonella serotyping is an expensive and time consuming process that requires implementing a battery of O and H antisera to detect 2,541 different Salmonella enterica serovars. For these reasons, we developed a rapid multiplex polymerase chain reaction (PCR)-based typing scheme to screen for the prevalent S. enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.     

The missense variation landscape of FTO,MC4R,and TMEM18 in obese children of African Ancestry

Objective:

Common variation at the loci harboring fat mass and obesity (FTO), melanocortin receptor 4 (MC4R), and transmembrane protein 18 (TMEM18) is consistently reported as being statistically most strongly associated with obesity. Investigations if these loci also harbor rarer missense variants that confer substantially higher risk of common childhood obesity in African American (AA) children were conducted.

Design and Methods:

The exons of FTO, MC4R, and TMEM18 in an initial subset of our cohort were sequenced, that is, 200 obese (BMI≥95th percentile) and 200 lean AA children (BMI≤5th percentile). Any missense exonic variants that were uncovered went on to be further genotyped in a further 768 obese and 768 lean (BMI≤50th percentile) children of the same ethnicity.

Results:

A number of exonic variants were observed from our sequencing effort: seven in FTO, of which four were non‐synonymous (A163T, G182A, M400V, and A405V), thirteen in MC4R, of which six were non‐synonymous (V103I, N123S, S136A, F202L, N240S, and I251L), and four in TMEM18, of which two were non‐synonymous (P2S and V113L). Follow‐up genotyping of these missense variants revealed only one significant difference in allele frequency between cases and controls, namely with N240S in MC4R (Fisher's exact P = 0.0001).

Conclusion:

In summary, moderately rare missense variants within the FTO, MC4R, and TMEM18 genes observed in our study did not confer risk of common childhood obesity in African Americans except for a degree of evidence for one known loss‐of‐function variant in MC4R.     

The effect of time of day and exercise on platelet functions and platelet–neutrophil aggregates in healthy male subjects

Platelet activation state changes by exercise. The effect of exercise time on platelet activation state and formation of platelet–neutrophil aggregates are not known yet. In this study the effect of exercise and time of day were examined on platelet activity with platelet–neutrophil aggregates. Ten moderately active males aged 27± 1.63 (mean±S.D.) years completed sub-maximal (70% VO_2max) exercise trials for 30 min. Blood pressure (BP) was recorded. Venous blood samples were obtained at rest, immediately post-exercise and after 30 min of recovery. Whole blood was analysed for haematocrit (Hct), haemoglobin (Hb), platelet count (PC), mean platelet count (MPV) and platelet aggregation (PA). Platelet–neutrophil aggregates and beta-thromboglobulin (β-TG) levels were assayed. Platelet count showed significant increase after morning exercise ((236± 32)×10⁹ l⁻¹ versus (202± 34)×10⁹ l⁻¹ baseline, p < 0.05). Exercise resulted in significantly lower MPV after the evening exercise (9.16± 0.5 fl versus 9.65± 0.36 fl, p < 0.05). Platelet aggregation by adenosine diphosphate (ADP) decreased after morning exercise and the recovery aggregation levels were significantly different at two different times of the day (68± 20% a.m. versus 80± 12% p.m., p < 0.05). It was also showed that platelet–neutrophil aggregates increased significantly from baseline after both exercises. Exercise-induced platelet–neutrophil aggregates were higher in the evening (10.7± 1.3% p.m. versus 6.4± 1.8% a.m., p < 0.0001). It is therefore concluded that besides platelet–platelet aggregation, exercise can cause platelet– neutrophil aggregates. In addition, time of day has an effect on platelet activation related events. Circadian variations of physiological parameters may have an effect on thrombus formation by platelet activation. (Mol Cell Biochem xxx: 119–124, 2005)     

Staphylococcal alpha‐phenol soluble modulins contribute to neutrophil lysis after phagocytosis

Staphylococcus aureus community‐acquired (CA) MRSA strains are highly virulent and can cause infections in otherwise healthy individuals. The most important mechanism of the host for clearing S. aureus is phagocytosis by neutrophils and subsequent killing of the pathogen. Especially CA‐MRSA strains are very efficient in circumventing this neutrophil killing. Interestingly, only a relative small number of virulence factors have been associated with CA‐MRSA, one of which are the phenol soluble modulins (PSMs). We have recently shown that the PSMs are functionally inhibited by serum lipoproteins, indicating that PSMs may exert their cytolytic function primarily in the intracellular environment. To further investigate the intracellular role of the PSMs we measured the effect of the α‐type and β‐type PSMs on neutrophil killing after phagocytosis. Using fluorescently labelled S. aureus, we measured bacterial survival after phagocytosis in a plate reader, which was employed next to flow cytometry and time‐lapse microscopy. Phagocytosis of the CA‐MRSA strain MW2 by human neutrophils resulted in rapid host cell death. Using mutant strains of MW2, we demonstrated that in the presence of serum, the intracellular expression of only the psmα operon is both necessary and sufficient for both increasedneutrophil cell death and increased survival of S. aureus. Our results identify PSMα peptides as prominent contributors to killing of neutrophils after phagocytosis, a finding with major implications for our understanding of S. aureus pathogenesis and strategies for S. aureus vaccine development.     

A New Methodology for Differentiation and Typing of Closely Related Salmonella enterica serovar Heidelberg Isolates

This study describe the use of a combination of two recently proposed typing approaches, multiple amplification of prophage locus typing (MAPLT) and multiple-locus variable-number tandem-repeat analysis (MLVA) for subdividing within Salmonella enterica serovar Heidelberg (S. Heidelberg). The combined typing method was compared with pulsed-field gel electrophoresis (PFGE) by Simpson’s index of diversity (DI). PFGE was shown to have a DI?=?0.84 and was poor at differentiation of the predominant PT1 (Phage Type 1) phenotype. In comparison, the combined MAPLT/MLVA method comprising 3 MLVA and 9 MAPLT primer pairs provided a higher differentiating ability DI?=?0.92. More importantly, the combined methodology was found to be superior in the differentiation of the predominant PT1 isolates. In conclusion, this study demonstrated the potential of the rapid and simple amalgamated MAPLT/MLVA approach in determining transmission of isolates of clonal phage type groups from various environmental sources to humans.     

Construction of a fusion flagellin complex and evaluation of the protective immunity of it in red snapper (Lutjanus sanguineus)

Aims: The main aims of this study were to construct a bivalent subunit vaccine containing flagellin flaA gene and flagellin flaB gene from Vibrio alginolyticus strain HY9901 and to explore the potential application of the fusion protein FlaA‐(G₄S)₃‐FlaB as a vaccine candidate for red snapper (Lutjanus sanguineus). Methods and Results: Flagellin gene flaA and flaB of V. alginolyticus were linked by gene SOEing (gene splicing by overlap extension) technology. The expression of the fusion gene flaA‐(G₄S)₃‐flaB in Escherichia coli BL21(DE3) was confirmed by SDS‐PAGE. Western blot analysis showed that the fusion protein FlaA‐(G₄S)₃‐FlaB, which was purified by affinity chromatography on Ni‐NTA resin, had positive reaction with mouse anti‐FlaA serum and mouse anti‐FlaB serum, respectively. The immunoprotection of FlaA‐(G₄S)₃‐FlaB as a bivalent subunit vaccine was investigated in red snapper model by enzyme‐linked immunosorbent assay (ELISA) and challenge test. Red snapper vaccinated with FlaA‐(G₄S)₃‐FlaB produced specific antibodies and were highly resistant to infection by virulent V. alginolyticus. Conclusions: The fusion gene flaA‐(G₄S)₃‐flaB from V. alginolyticus strain HY9901 was cloned by gene SOEing and was expressed in E. coli. This fusion protein FlaA‐(G₄S)₃‐FlaB is a good protective antigen of V. alginolyticus and should be considered as an effective vaccine candidate against infection by V. alginolyticus in red snapper. Significance and Impact of the Study: Two flagellin genes, flaA and flaB, which are independent in structure and function, were first linked together by gene SOEing technology. The finding that red snapper did adequately respond to the fusion protein FlaA‐(G₄S)₃‐FlaB injection made it a promising candidate for vaccine treatment. To develop effective vaccine candidates against V. alginolyticus, more attention should be given to these immunogenic flagellins.     

Molecular cloning and characterization of the genetic determinants that express the complete Shigella serotype D (Shigella sonnei) lipopolysaccharide in heterologous live attenuated vaccine strains

The genetic determinants for the complete Shigella sonnei lipopolysaccharide (LPS) have been cloned, characterized by restriction mapping, and expressed in heterologous genetic backgrounds, including Salmonella typhi and Vibrio cholerae live attenuated vaccine strains. The rfb/rfc locus encoding the polymerized serotype-specific O polysaccharide was mapped within 23 kb of DNA isolated from S. sonnei virulence plasmid pWR105. A highly similar chromosomal DNA sequence was identified by Southern hybridization analysis in Plesiomonas shigelloides known to have the same O serotype specificity as S. sonnei. Expression studies of the rfb/rfc locus have shown that S. sonnei. O polysaccharide is covalently bound to LPS cores of both the K-12 and RI types, but neither to Salmonella (Ra-type) nor to V. cholerae O1 cores. In order to express a compatible core structure in the latter organisms, chromosomal rfa loci encoding R1-type LPS were isolated from both an Escherichia coli R1 strain (rfaR1) and from S. sonnei (rfd_sonnei). Restriction mapping and functional analysis of cloned DNA allowed us to localize the rfaR1 locus and to orient it with respect to the neighbouring cysE chromosomal marker. A high degree of sequence similarity was found at the DNA level between rfa loci of enterobacterial species characterized by Ri-type LPS. Co-expression studies involving S. sonnei rfb/rfc and rfa loci propagated on compatible plasmids have shown that, at most, 13 to 14 kb of r/api DNA are required for the expression of complete phase-l-like S. sonnei LPS in E. coli K-12 and S. typhi, whereas an adjacent region of about 3.5 kb is needed in the more stringent host, V. cholerae, S. sonnei O antigen expressed in a V. eholerae recombinant vaccine strain is present on the cell surface in a form suitable for the induction of a specific antibody response in vaccinated rabbits.     

Tracing Streptococcus thermophilus strains in three-component yogurt starters

Three-component starters for yogurt were obtained on the base of starter LBB.BY 5-12 for traditional Bulgarian yogurt, containing strains Lactobacillus delbrueckii ssp. bulgaricus B5 and Streptococcus thermophilus A with the addition of either an exopolysaccharide-producing S. thermophilus strain 6V or the fast acidifying S. thermophilus strain N1. To differentiate between the three strains in the starter cultures, randomly amplified polymorphic DNA (RAPD) technique was applied to develop strain-specific probes. Southern hybridization against dot-blots of chromosomal DNA from the three S. thermophilus strains confirmed that two probes, derived from a 770 bp RAPD product obtained with primer RAPD-4 and a 290 bp sequence obtained with primer OPP-7 were specific for S. thermophilus 6V and S. thermophilus A, respectively, while no hybridization to S. thermophilus N1 DNA was observed. The selected probes were used to differentiate between S. thermophilus colonies on a solid agar medium by colony hybridization. The evaluation of the viable cell counts revealed that the populations of S. thermophilus A and the added S. thermophilus strains 6V or N1 in the three-component starters and in yogurt had nearly equal proportion allowing each strain to contribute to the enriched properties of starter and product.     

Molecular cytogenetic characterisation and phylogenetic analysis of the seven cultivated Vigna species (Fabaceae)

The genomic organisation of the seven cultivated Vigna species, V. unguiculata, V. subterranea, V. angularis, V. umbellata, V. radiata, V. mungo and V. aconitifolia, was determined using sequential combined PI and DAPI (CPD) staining and dual‐colour fluorescence in situ hybridisation (FISH) with 5S and 45S rDNA probes. For phylogenetic analyses, comparative genomic in situ hybridisation (cGISH) onto somatic chromosomes and sequence analysis of the internal transcribed spacer (ITS) of 45S rDNA were used. Quantitative karyotypes were established using chromosome measurements, fluorochrome bands and rDNA FISH signals. All species had symmetrical karyotypes composed of only metacentric or metacentric and submetacentric chromosomes. Distinct heterochromatin differentiation was revealed by CPD staining and DAPI counterstaining after FISH. The rDNA sites among all species differed in their number, location and size. cGISH of V. umbellata genomic DNA to the chromosomes of all species produced strong signals in all centromeric regions of V. umbellata and V. angularis, weak signals in all pericentromeric regions of V. aconitifolia, and CPD‐banded proximal regions of V. mungo var. mungo. Molecular phylogenetic trees showed that V. angularis and V. umbellata were the closest relatives, and V. mungo and V. aconitifolia were relatively closely related; these species formed a group that was separated from another group comprising V. radiata, V. unguiculata ssp. sesquipedalis and V. subterranea. This result was consistent with the phylogenetic relationships inferred from the heterochromatin and cGISH patterns; thus, fluorochrome banding and cGISH are efficient tools for the phylogenetic analysis of Vigna species.     

Ricin A Chain from <Emphasis Type="Italic">Ricinus sanguineus</Emphasis>: DNA Sequence,Structure and Toxicity

The gene coding for the ricin A-chain from Ricinus sanguineus (R_sTA) was isolated and sequenced (GB: DQ661048). Comparison of R_sTA with the ricin A-chain from Ricinus communis (RTA) revealed the presence of five differences in the gene sequence. At the protein level only two differences were noticed, the replacement of Asn136 by Ser (N136S) and Ile173 by Val (I174 V). From the R_sTA structure model (PMDB: PM0074652), the N136S mutation was predicted to have no effect on R_sTA structure. The I174V mutation is believed to have no effect on the R_sTA structure or on toxicity since this replacement was found in the Ricinus agglutinin’s A-chain and the latter has a comparable toxicity to RTA. The Ser221 of the putative lipase active site was found. Toxicity experiments showed that R_sTA and RTA have similar toxicities. This finding proves that the N136S and I174 V mutations have no effect on R_sTA toxicity.