The Karyotype of <Emphasis Type="Italic">Amoeba borokensis</Emphasis> from a “<Emphasis Type="Italic">proteus</Emphasis>-like” Amoeba Group (Amoebozoa: Euamoebida)

The karyotype of Amoeba borokensis was studied for the first time. At the metaphase of mitosis, this species has a haploid set of chromosomes (n = 27). This is exactly half of the diploid karyotype in Amoeba proteus (strain B). At first glance, it confirms the idea that has recently appeared that one species arises from another via multiple changes in chromosome number. However, comparative cytogenetic analysis revealed that four orthologous chromosomes from haploid sets of these two species were not distinguished by the pattern of DAPI-stained bands. It shows that the genetic distance between A. proteus and A. borokensis is higher than was believed earlier and these two species have diverged from each other. Analysis of data on the life cycles of A. proteus and A. borokensis shows that a kind of so-called “cyclic polyploidy” takes place in “proteus-like” amoebae. “Cyclic polyploidy” has recently been considered as an alternative to the sexual process for genetic recombination in agamic protists from different macrotaxons.     

Optical tomography analysis of <Emphasis Type="Italic">Amoeba proteus</Emphasis> chromatin organization at various cell cycle stages

An optical tomography investigation of the nuclear cycle in large freshwater amoebae Amoeba proteus has been performed for the first time. Nuclei of cells from a synchronized culture were stained with DAPI and examined using a confocal laser scanning microscope. Detailed analysis of three-dimensional images of the intranuclear chromatin at different stages of the nuclear cycle has been performed. The materials obtained, in combination with the published data, allow for a completely new representation of the dynamics of the structural organization of the A. proteus nucleus during the cell cycle. Two-stage interphase and mitosis of a special type not matching any of the known types in the existing systems of classification of mitosis were found to occur in amoebae. Amplification of chromosomes and/or fragments thereof supposedly occurs during the cell cycle, which is consistent with the available data on nuclear DNA hyperreplication during the cell cycle of A. proteus. The number of chromosomes can vary at different stages of the cycle because of amplification, this being a putative reason for the discordant reports on the number of chromosomes in this species. The elimination of “excess” DNA mainly occurs during the transition from prophase to prometaphase. Finally, specific features of chromosome behavior during mitosis allow conclusion to be drawn that many, if not all, chromosomes are of a holocentric type.     

A New Recombinant Strain of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Producing Encapsulated Phytase from <Emphasis Type="Italic">Obesumbacterium proteus</Emphasis>

A new recombinant strain of Yarrowia lipolytica synthesizing encapsulated highly thermostable phytase of Obesumbacterium proteus, which is recommended for use as a premix component of feed compositions in animal husbandry, was obtained.     

On the multicomponent nature of <Emphasis Type="Italic">Halobacterium salinarum</Emphasis> flagella

Filaments of the flagellum of the halophilic archaeon Halobacterium salinarum consist of five flagellins: A1, A2, B1, B2, and B3, which are encoded by five genes localized in tandem in two flgA and flgB operons. While the role of flagellins A1 and A2 has been determined, the role of the proteins, B operon products, is still unclear. A mutant strain of H. salinarum with deleted A and B flagellin genes (ΔflgAΔflgB) has been obtained for the first time. This strain has been used to create and analyze the strains carrying only individual B1 or B3 flagellin genes. Cells of the ΔflgAΔflgB strain were shown to have short filamentous formations, 7–8 nm thick, which we have named as X-filaments. It has been shown that X-filaments consist of a protein immunologically related to flagellins A and B. Expression of the B1 and B3 genes is suppressed in the absence of A1, A2, and B2. It has been shown that flagellins B1 and B3 cannot be substituted for flagellin B2 upon the formation of a curved hook-like structure, which serves as a connecting element between the flagellar filament and the motor axis. The multicomponent nature of flagella is discussed in the light of their possible involvement in other cell processes besides providing motility.     

B-chromosome frequency stability in <Emphasis Type="Italic">Prochilodus lineatus</Emphasis> (Characiformes,Prochilodontidae)

The genus Prochilodus includes individuals ranging in size from medium to large, being highly relevant for commercial and subsistence fishing. Prochilodus species have a diploid number of 2n = 54 chromosomes and up to seven supernumerary (B) microchromosomes. Previous research has shown that B frequency increased drastically in the Mogi-Guaçu river population of Prochilodus lineatus in the early 1980s, whereas it remained about constant in the 1990s. Here we analyses B frequency in this population during the 2003–2007 period and have found that frequency has not changed significantly since 1987, and that these B chromosomes do not show the intra-individual variation in number that characterized them in the 1980s. This indicates that these B chromosomes have been neutralized, after their invasion, through their mitotic stabilization.     

Centromeric and non-centromeric satellite DNA organisation differs in holocentric <Emphasis Type="Italic">Rhynchospora</Emphasis> species

Satellite DNA repeats (or satDNA) are fast-evolving sequences usually associated with condensed heterochromatin. To test whether the chromosomal organisation of centromeric and non-centromeric satDNA differs in species with holocentric chromosomes, we identified and characterised the major satDNA families in the holocentric Cyperaceae species Rhynchospora ciliata (2n = 10), R. globosa (2n = 50) and R. tenuis (2n = 2x = 4 and 2n = 4x = 8). While conserved centromeric repeats (present in R. ciliata and R. tenuis) revealed linear signals at both chromatids, non-centromeric, species-specific satDNAs formed distinct clusters along the chromosomes. Colocalisation of both repeat types resulted in a ladder-like hybridisation pattern at mitotic chromosomes. In interphase, the centromeric satDNA was dispersed while non-centromeric satDNA clustered and partly colocalised to chromocentres. Despite the banding-like hybridisation patterns of the clustered satDNA, the identification of chromosome pairs was impaired due to the irregular hybridisation patterns of the homologues in R. tenuis and R. ciliata. These differences are probably caused by restricted or impaired meiotic recombination as reported for R. tenuis, or alternatively by complex chromosome rearrangements or unequal condensation of homologous metaphase chromosomes. Thus, holocentricity influences the chromosomal organisation leading to differences in the distribution patterns and condensation dynamics of centromeric and non-centromeric satDNA.     

Molecular characterization of <Emphasis Type="Italic">bmyC</Emphasis> gene of the mosquito pupicidal bacteria, <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> (VCRC B483) and in silico analysis of bacillomycin D synthetase C protein

A strain of Bacillus amyloliquefaciens (VCRC B483) exhibiting mosquito pupicidal, keratinase and antimicrobial activities was isolated from mangrove forest ecosystem of Andaman and Nicobar Islands. Molecular characterization of the strain showed the presence of lipopeptide encoding bmyC gene. Phylogenetic tree based on protein sequence of this gene exhibited homology with mycosubtilin synthetase of Bacilus atropheus and Iturin synthetase of Bacillus subtilis and B. amyloliquefaciens. This is the first report on the evolutionary conservation of amino acids concerned with the function and structure of bmyC protein of B. amyloliquefaciens. The presence of valine at the 1197th position in our strain was found to be unique and different from the existing strains of B. subtilis and B. amyloliquefaciens. Molecular modelling studies revealed significant changes in the structure of epimerization domain of the bmyC protein with A1197V variation. Crude metabolite of this strain exhibited antifungal activity against Fusarium sp. and Carvularia sp.     

Quinacrine Fluorescence Patterns and Terminal DNA Labelling of Human <Emphasis Type="Italic">C</Emphasis> Group Chromosomes

THE human C group chromosomes have been difficult to study because they have rather similar morphology. Application of the quinacrine fluorescent staining technique developed by Caspersson et al.¹ now allows the identification of individual chromosomes and of the chromosome segments involved in translocations because the fluorescent patterns are not altered by the translocation^2–4. We have reported the value of this technique in analysing abnormalities of the D⁴ and G³ groups. We report here a variety of structural changes of C group chromosomes which have been characterized in this way, as well as the terminal DNA replication pattern of the C group chromosomes.     

Molecular and cytological analyses of A and C genomes at meiosis in synthetic allotriploid <Emphasis Type="Italic">Brassica</Emphasis> hybrids (ACC) between <Emphasis Type="Italic">B.napus</Emphasis> (AACC) and <Emphasis Type="Italic">B.oleracea</Emphasis> (CC)

Success of interspecific hybridization relies mostly on the adequate similarity between the implicated genomes to ensure synapsis, pairing and recombination between appropriate chromosomes during meiosis in allopolyploid species. Allotetraploid Brassica napus (AACC) is a model of natural hybridization between Brassica rapa (AA) and Brassica oleracea (CC), which are originally derived from a common ancestor, but genomic constitution of the same chromosomes probably varied among these species through time after establishment, giving rise to cytogenetic difference in the synthetic hybrids. Herein we investigated meiotic behaviors of A and C chromosomes of synthetic allotriploid Brassica hybrids (ACC) at molecular and cytological levels, which result from the interspecific cross between natural B. napus (AACC) and B.oleracea (CC), and the results showed that meiosis course was significantly aberrant in allotriploid Brassica hybrids, and chromosomes aligned chaotically at metaphase I, chromosome bridges and lags were frequently observed from later metaphase I to anaphase II during meiosis. Simultaneously, we also noticed that meiosis-related genes were abruptly down-regulated in allotriploid Brassica hybrids, which likely accounted for irregular scenario of meiosis observed in these synthetic hybrids. Therefore, these results indicated that inter-genomic exchanges of A and C chromosomes could occur frequently in synthetic Brassica hybrids, and provided an efficient approach for genetic changes of homeologous chromosomes during meiosis in polyploid B.napus breeding program.     

Genome-wide characterization and stress-responsive expression profiling of <Emphasis Type="Italic">MCM</Emphasis> genes in <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">Brassica rapa</Emphasis>

The Minichromosome maintenance protein [MCM (2-7)] complex is associated with helicase activity for replication fork formation during DNA replication. We identified and characterized each 12 putative MCM genes from Brassica oleracea and Brassica rapa. MCM genes were classified into nine groups according to their evolutionary relationships. A high number of syntenic regions were present on chromosomes C03 and A03 in B. oleracea and B. rapa, respectively, compared to the other chromosomes. Expression analysis showed that most of the MCM(2-7) helicase-subunit genes and their coregulating MCM genes were upregulated during hydroxyurea (HU) induced stress in B. oleracea. In B. rapa, MCM(2-7) helicase genes BrMCM2_2, BrMCM7_1, BrMCM7_2 and their co-regulating genes were upregulated during replication stress. During cold stress, BoMCM6 in B. oleracea and BrMCM5 in B. rapa were remarkably upregulated. During salt stress, BoMCM6_2, BoMCM7_1, BoMCM8, BoMCM9, and BoMCM10 were markedly upregulated in B. oleracea. Hence, our study identified the candidate MCM family genes those possess abiotic stress-responsive behavior and DNA replication stress tolerance. As the first genome-wide analysis of MCM genes in B. oleracea and B. rapa, this work provides a foundation to develop stress responsive plants. Further functional and molecular studies on MCM genes will be helpful to enhance stress tolerance in plants.     

Characterization of <Emphasis Type="Italic">lpaH2</Emphasis> gene corresponding to lipopeptide synthesis in <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> HAB-2

Background

Bacillus spp. have prominent ability to suppress plant pathogens and corresponding diseases. Previous analyses of Bacillus spp. revealed numerous gene clusters involved in nonribosomal synthesis of cyclic lipopeptides with distinct antimicrobial action. The 4′-phosphopantetheinyl transferase (PPTase) encoded by sfp gene is a key factor in lipopeptide synthesis in Bacillus spp. In previous study, B. amyloliquefaciens strain HAB-2 was found to inhibit a broad range of plant pathogens, which was attributed to its secondary metabolite lipopeptide.

Results

A sfp homologue lpaH2 which encoded phosphopantetheinyl transferase but shared 71% sequence similarity was detected in strain HAB-2. Disruption of lpaH2 gene resulted in losing the ability of strain HAB-2 to produce lipopeptide, as well as antifungal and hemolytic activities. When lpaH2 replaced sfp gene of B. subtilis strain 168, a non-lipopeptide producer, the genetically engineered strain 168 could produced lipopeptides and recovered antifungal activity. Quantitative PCR assays indicated that, the expression level of lpaH2 in B. subtilis 168 strain decrease to 0.27-fold compared that of the wild type B. amyloliquefaciens strain HAB-2.

Conclusion

Few studies have reported about lpa gene which can replace sfp gene in the different species. Taken together, our study showed for the first time that lpaH2 from B. amyloliquefaciens could replace sfp gene.

     

Development,genetic and cytogenetic analyses of genetic sexing strains of the Mexican fruit fly, <Emphasis Type="Italic">Anastrepha ludens</Emphasis> Loew (Diptera: Tephritidae)

Background

Anastrepha ludens is among the pests that have a major impact on México's economy because it attacks fruits as citrus and mangoes. The Mexican Federal government uses integrated pest management to control A. ludens through the Programa Nacional Moscas de la Fruta [National Fruit Fly Program, SAGARPA-SENASICA]. One of the main components of this program is the sterile insect technique (SIT), which is used to control field populations of the pest by releasing sterile flies.

Results

To increase the efficiency of this technique, we have developed a genetic sexing strain (GSS) in which the sexing mechanism is based on a pupal colour dimorphism (brown-black) and is the result of a reciprocal translocation between the Y chromosome and the autosome bearing the black pupae (bp) locus. Ten strains producing wild-type (brown pupae) males and mutant (black pupae) females were isolated. Subsequent evaluations for several generations were performed in most of these strains. The translocation strain named Tapachula-7 showed minimal effect on survival and the best genetic stability of all ten strains. Genetic and cytogenetic analyses were performed using mitotic and polytene chromosomes and we succeeded to characterize the chromosomal structure of this reciprocal translocation and map the autosome breakpoint, despite the fact that the Y chromosome is not visible in polytene nuclei following standard staining.

Conclusions

We show that mitotic and polytene chromosomes can be used in cytogenetic analyses towards the development of genetic control methods in this pest species. The present work is the first report of the construction of GSS of Anastrepha ludens, with potential use in a future Moscafrut operational program.

     

Cytogenetic characterization of <Emphasis Type="Italic">Amaranthus caudatus</Emphasis> L. and <Emphasis Type="Italic">Amaranthus hybridus</Emphasis> subsp<Emphasis Type="Italic">. cruentus</Emphasis> (L.) Thell.

The present study is aimed to identify genetic variability between two species of Amaranthus viz., A. caudatus and A. hybridus subsp. cruentus, two economically important species, cultivated mainly for grain production. Karyomorphological studies in Amaranthus are scarce, probably due to higher number of small sized chromosomes. Karyomorphological studies were conducted using mitotic squash preparation of young healthy root tips. Karyological parameters and karyotypic formula were established using various software programs and tabulated the karyomorphometric and asymmetry indices viz., Disparity index, Variation coefficient, Total forma percentage, Karyotype asymmetry index, Syi index, Rec index, Interchromosomal and Intrachromosomal asymmetry index and Degree of asymmetry of karyotypes. The mitotic chromosome number observed for A. caudatus was 2n = 32 with a gametic number n = 16 and A. hybridus subsp. cruentus was 2n = 34 with a gametic number n = 17. In A. caudatus the chromosome length during somatic metaphase ranged from 0.8698 to 1.7722 μm with a total length of 39.1412 μm. In A. hybridus subsp. cruentus the length of chromosome ranged from 0.7756 to 1.9421 μm with a total length of 44.9922 μm. Various karyomorphometry and asymmetry indices analyzed revealed the extend of interspecific variation and their evolutionary status.     

<Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> isolated from cheese: production and partial characterization of bacteriocin B391

Lactobacillus plantarum B391, a strain isolated from an artisanal French cheese, is a producer of a bacteriocin, expressing activity against Enterococcus faecalis NCTC 775, Clostridium perfringens NCTC 13170 and several Listeria monocytogenes strains. High stability was recorded after heat treatment at 121 °C for 20 min and when stored at 4 °C for more than 40 days. A challenge test performed in milk for 11 days showed potential for the control of L. monocytogenes. In the presence of the lytic bacteriocin B391, L. monocytogenes cells present numerous morphology modifications of cell shape and surface structure as well as in the cell division pattern, resulting ultimately in lysis. The high level of Listeria growth inhibition obtained in the presence of Lb. plantarum B391, and the stability of B391 bacteriocin for a long period of time, make this strain potentially interesting to use in milk products to increase food safety.     

The effect of intercultivar substitution of wheat <Emphasis Type="Italic">Triticum aestivum</Emphasis> L. chromosomes on lipoxygenase activity and its correlation with the technological properties of flour

The effects of intercultivar substitution of individual chromosome pairs (except for 1B, 6D, and 7A) in the wheat cultivars Saratovskaya 29 and Janetzkis Probat, differing in quality, on specific lipoxygenase activity, the grain yield per spike, and the main technological properties of flour and dough were studied. It was demonstrated that the substitution of individual chromosomes of the recipient cultivar Saratovskaya 29 with the homologous chromosomes of the donor cultivar Janetzkis Probat caused significant changes in lipoxygenase activity and several other quality characteristics. The correlations between the lipoxygenase activity and the parameters of physical dough properties were determined. Three molecular forms of lipoxygenase (Lpx-1, Lpx-2, and Lpx-3), differing in the value of surface charge and enzymatic activity, were detected by native PAGE.     

Development of a genetic sexing strain in <Emphasis Type="Italic">Bactrocera carambolae</Emphasis> (Diptera: Tephritidae) by introgression of sex sorting components from <Emphasis Type="Italic">B. dorsalis</Emphasis>, Salaya1 strain

Background

The carambola fruit fly, Bactrocera carambolae Drew & Hancock is a high profile key pest that is widely distributed in the southwestern ASEAN region. In addition, it has trans-continentally invaded Suriname, where it has been expanding east and southward since 1975. This fruit fly belongs to Bactrocera dorsalis species complex. The development and application of a genetic sexing strain (Salaya1) of B. dorsalis sensu stricto (s.s.) (Hendel) for the sterile insect technique (SIT) has improved the fruit fly control. However, matings between B. dorsalis s.s. and B. carambolae are incompatible, which hinder the application of the Salaya1 strain to control the carambola fruit fly. To solve this problem, we introduced genetic sexing components from the Salaya1 strain into the B. carambolae genome by interspecific hybridization.

Results

Morphological characteristics, mating competitiveness, male pheromone profiles, and genetic relationships revealed consistencies that helped to distinguish Salaya1 and B. carambolae strains. A Y-autosome translocation linking the dominant wild-type allele of white pupae gene and a free autosome carrying a recessive white pupae homologue from the Salaya1 strain were introgressed into the gene pool of B. carambolae. A panel of Y-pseudo-linked microsatellite loci of the Salaya1 strain served as markers for the introgression experiments. This resulted in a newly derived genetic sexing strain called Salaya5, with morphological characteristics corresponding to B. carambolae. The rectal gland pheromone profile of Salaya5 males also contained a distinctive component of B. carambolae. Microsatellite DNA analyses confirmed the close genetic relationships between the Salaya5 strain and wild B. carambolae populations. Further experiments showed that the sterile males of Salaya5 can compete with wild males for mating with wild females in field cage conditions.

Conclusions

Introgression of sex sorting components from the Salaya1 strain to a closely related B. carambolae strain generated a new genetic sexing strain, Salaya5. Morphology-based taxonomic characteristics, distinctive pheromone components, microsatellite DNA markers, genetic relationships, and mating competitiveness provided parental baseline data and validation tools for the new strain. The Salaya5 strain shows a close similarity with those features in the wild B. carambolae strain. In addition, mating competitiveness tests suggested that Salaya5 has a potential to be used in B. carambolae SIT programs based on male-only releases.

     

The <Emphasis Type="Italic">Agropyron cristatum</Emphasis> karyotype,chromosome structure and cross-genome homoeology as revealed by fluorescence in situ hybridization with tandem repeats and wheat single-gene probes

Key message

Fluorescence in situ hybridization with probes for 45 cDNAs and five tandem repeats revealed homoeologous relationships of Agropyron cristatum with wheat. The results will contribute to alien gene introgression in wheat improvement.

Abstract

Crested wheatgrass (Agropyron cristatum L. Gaertn.) is a wild relative of wheat and a promising source of novel genes for wheat improvement. To date, identification of A. cristatum chromosomes has not been possible, and its molecular karyotype has not been available. Furthermore, homoeologous relationship between the genomes of A. cristatum and wheat has not been determined. To develop chromosome-specific landmarks, A. cristatum genomic DNA was sequenced, and new tandem repeats were discovered. Their distribution on mitotic chromosomes was studied by fluorescence in situ hybridization (FISH), which revealed specific patterns for five repeats in addition to 5S and 45S ribosomal DNA and rye subtelomeric repeats pSc119.2 and pSc200. FISH with one tandem repeat together with 45S rDNA enabled identification of all A. cristatum chromosomes. To analyze the structure and cross-species homoeology of A. cristatum chromosomes with wheat, probes for 45 mapped wheat cDNAs covering all seven chromosome groups were localized by FISH. Thirty-four cDNAs hybridized to homoeologous chromosomes of A. cristatum, nine hybridized to homoeologous and non-homoeologous chromosomes, and two hybridized to unique positions on non-homoeologous chromosomes. FISH using single-gene probes revealed that the wheat-A. cristatum collinearity was distorted, and important structural rearrangements were observed for chromosomes 2P, 4P, 5P, 6P and 7P. Chromosomal inversions were found for pericentric region of 4P and whole chromosome arm 6PL. Furthermore, reciprocal translocations between 2PS and 4PL were detected. These results provide new insights into the genome evolution within Triticeae and will facilitate the use of crested wheatgrass in alien gene introgression into wheat.

     

Establishment of an efficient transformation system for <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

Pleurotus ostreatus is widely cultivated worldwide, but the lack of an efficient transformation system regarding its use restricts its genetic research. The present study developed an improved and efficient Agrobacterium tumefaciens-mediated transformation method in P. ostreatus. Four parameters were optimized to obtain the most efficient transformation method. The strain LBA4404 was the most suitable for the transformation of P. ostreatus. A bacteria-to-protoplast ratio of 100:1, an acetosyringone (AS) concentration of 0.1 mM, and 18 h of co-culture showed the best transformation efficiency. The hygromycin B phosphotransferase gene (HPH) was used as the selective marker, and EGFP was used as the reporter gene in this study. Southern blot analysis combined with EGFP fluorescence assay showed positive results, and mitotic stability assay showed that more than 75% transformants were stable after five generations. These results showed that our transformation method is effective and stable and may facilitate future genetic studies in P. ostreatus.     

Molecular cytogenetic analysis of DNA from pericentric heterochromatin of chromosome 2L of malaria mosquito <Emphasis Type="Italic">Anopheles beklemishevi</Emphasis> (culicidae,Diptera)

Using the method of microdissection of polytene chromosomes, followed by in situ hybridization, chromosomal localization of region-specific DNA probe from pericentic heterochromatin of chromosome 2L of Anopheles beklemishevi Stegnii et Kabanova was examined on polytene chromosomes of Anopheles atroparvus van Thiel, An. messeae Fall, and An. beklemishevi. DNA sequences homologous to the probe used were found in all species examined on chromosomes 2 and 3 in pericentric regions and in attachment regions. The exclusion were the attachment regions of chromosome XL in An. beklemishevi and An. messeae, and pericentric region of arm 2R in An. messeae. Pericentric α -heterochromatin of arm 2L in An. messeae and arm 3R in An. atroparvus also contained no sequences homologous to the DNA probe. The data obtained were compared with the earlier obtained data on localization of species-specific probe from the segment of chromosome 2R of An. atroparvus on chromosomes of An. artoparvus, An. messeae, and An. beklemishevi. The differences between the species in the sites of probes localization and fluorescence intensity revealed pointed to the existence of individual sequence associations in the regions of chromosomes attachment.