Non-transcribed Spacer Research Articles

Molecular strain typing of Trichophyton mentagrophytes (T. mentagrophytes var. interdigitale) using non-transcribed spacer region as a molecular marker.

Background & objectives:Dermatophytes are keratinophilic fungi that infect keratinized tissues of human and animal origin. Trichophyton mentagrophytes is considered to be a species complex composed of several strains, which include both anthropophiles and zoophiles. Accurate discrimination is critical for comprehensive understanding of the clinical and epidemiological implications of the genetic heterogeneity of this complex. Molecular strain typing renders an effective way to discriminate each strain. The objective of the study was to characterize T. mentagrophytes clinical isolates to sub-species level using molecular techniques and non-transcribed spacer (NTS) region as marker.Methods:Sixty four T. mentagrophytes clinical isolates were identified by phenotypic methods. These were subjected to polymerase chain reaction targeting three sub-repeat elements (SREs), TmiS0, TmiS1 and TmiS2 of the NTS region. Sequence analysis of internal transcribed spacer (ITS) region of different types was also done.Results:Strain-specific polymorphism was observed in all three loci. Totally, 13 different PCR types were obtained on combining all the three SREs loci. No variation was observed in the ITS region.Interpretation & conclusions:The study described the usefulness of molecular strain typing technique for the discrimination of the T. mentagrophytes isolates. This will help for the future explorations into the epidemiology of T. mentagrophytes and its complex.

Evolutionary relationship of three mitten crabs (Eriocheir sp) revealed by mitogenome and 5S ribosomal DNA analysis

To reveal the evolutionary relationship of three mitten crabs (Eriocheir sinensis, E. hepuensis, and E. japonica), complete mitogenomes and nuclear 5S rDNA sequences were analyzed. Sequencing revealed that the mitogenomes analyzed shared conserved organization of the coding and non-coding regions but genetic variation was identified. Among the three mitten crabs distinct tandem repeats were identified in the mitochondrial D-loop region. The 5S gene (5S rDNA) sequence was highly conserved across the three species, whereas non-transcribed spacer (NTS) region exhibit high levels of variation including insertions, deletions and point mutations. Cluster analysis suggested that the three mitten crabs had their own independent 5S rDNA sequence variation and evolutionary pattern. Both mitogenome and 5S rDNA sequence analysis revealed significant genetic variation across the mitten crabs species. Phylogenetic analysis using mitogenome and 5S rDNA sequences demonstrated that E. japonica was relatively more distant from E. sinensis and E. hepuensis. This study extended our previous knowledge and confirmed that the three mitten crabs are likely to be genetically differentiated species. In addition, our study also provided insights into the conservation of pure natural resources of E. sinensis, an important aquaculture species.

Site-Specific Fluorescence Dynamics To Probe Polar Arrest by Fob1 in Replication Fork Barrier Sequences.

Fob1 protein plays an important role in aging and maintains genomic stability by avoiding clashes between the replication and transcription machinery. It facilitates polar arrest by binding to replication fork barrier (RFB) sites, present within the nontranscribed spacer region of the ribosomal DNA. Here, we investigate the mechanism of unidirectional arrest by creating multiple prosthetic forks within the RFB, with fluorescent adenine analogue 2-aminopurine incorporated site-specifically in both the “permissible” and “nonpermissible” directions. The motional dynamics of the RFB-Fob1 complexes analyzed by fluorescence lifetime and fluorescence anisotropy decay kinetics shows that Fob1 adopts a clamp-lock model of arrest and causes stronger perturbation with the bases in the double-stranded region of the nonpermissible-directed forks over those of the permissible directed ones, thereby creating a polar barrier. Corroborative thermal melting studies reveal a skewed distribution of GC content within the RFB sequence that potentially assists in Fob1-mediated arrest.

Molecular cytogenetic characterization and comparison of the two cultivated Canavalia species (Fabaceae).

The two cultivated Canavalia (Adanson, 1763) species, Canavalia gladiata (N. J. von Jacquin, 1788) A. P. de Candolle, 1825 and Canavalia ensiformis (Linnaeus, 1753) A. P. de Candolle, 1825 are closely related based on morphological and molecular phylogenetic data. However, the similarities and differences in genome organization between them have not been evaluated at molecular cytogenetic level. Here, detailed karyotypes of both species were constructed using combined PI and DAPI (CPD) staining, rDNA-FISH and self-genomic in situ hybridization (sGISH). For further comparison, comparative genomic in situ hybridization (cGISH) and sequence analysis of 5S rDNA were applied. Their chromosomes were accurately identified by sGISH and rDNA-FISH signals. Both species had the karyotype formula 2n = 22 = 18m + 4m-SAT, but the karyotype of C. ensiformis was shorter and more asymmetric than that of C. gladiata. They displayed similar CPD bands at all 45S rDNA sites and centromeres. C. gladiata had ten centromeric 5S rDNA loci and two SC (secondary constriction)-associated 45S rDNA loci. C. ensiformis had nine centromeric and one interstitial 5S loci, two SC-associated and one proximal 45S loci. Their sGISH signal patterns displayed both basic similarities and distinct differences. Reciprocal cGISH generated prominent signals in all pericentromeric regions and 45S sites. There was lower level of sequence identity of the non-transcribed spacer between their 5S rDNA repeats. These data confirmed the evolutionary closeness between C. gladiata and C. ensiformis and demonstrated obvious differentiation between their genomes, and supported the opinion that C. ensiformis is more advanced in evolution than C. gladiata.

Variations in 5S rDNAs in diploid and tetraploid offspring of red crucian carp \xd7 common carp

BackgroundThe allotetraploid hybrid fish (4nAT) that was created in a previous study through an intergeneric cross between red crucian carp (Carassius auratus red var., ♀) and common carp (Cyprinus carpio L., ♂) provided an excellent platform to investigate the effect of hybridization and polyploidization on the evolution of 5S rDNA. The 5S rDNAs of paternal common carp were made up of a coding sequence (CDS) and a non-transcribed spacer (NTS) unit, and while the 5S rDNAs of maternal red crucian carp contained a CDS and a NTS unit, they also contained a variable number of interposed regions (IPRs). The CDSs of the 5S rDNAs in both parental fishes were conserved, while their NTS units seemed to have been subjected to rapid evolution.ResultsThe diploid hybrid 2nF1 inherited all the types of 5S rDNAs in both progenitors and there were no signs of homeologous recombination in the 5S rDNAs of 2nF1 by sequencing of PCR products. We obtained two segments of 5S rDNA with a total length of 16,457 bp from allotetraploid offspring 4nAT through bacterial artificial chromosome (BAC) sequencing. Using this sequence together with the 5S rDNA sequences amplified from the genomic DNA of 4nAT, we deduced that the 5S rDNAs of 4nAT might be inherited from the maternal progenitor red crucian carp. Additionally, the IPRs in the 5S rDNAs of 4nAT contained A-repeats and TA-repeats, which was not the case for the IPRs in the 5S rDNAs of 2nF1. We also detected two signals of a 200-bp fragment of 5S rDNA in the chromosomes of parental progenitors and hybrid progenies by fluorescence in situ hybridization (FISH).ConclusionsWe deduced that during the evolution of 5S rDNAs in different ploidy hybrid fishes, interlocus gene conversion events and tandem repeat insertion events might occurred in the process of polyploidization. This study provided new insights into the relationship among the evolution of 5S rDNAs, hybridization and polyploidization, which were significant in clarifying the genome evolution of polyploid fish.

Genetic signature analysis of Perkinsus marinus in Mexico suggests possible translocation from the Atlantic Ocean to the Pacific coast of Mexico

BackgroundThe protozoan Perkinsus marinus (Mackin, Owen & Collier) Levine, 1978 causes perkinsosis in the American oyster Crassostrea virginica Gmelin, 1791. This pathogen is present in cultured C. virginica from the Gulf of Mexico and has been reported recently in Saccostrea palmula (Carpenter, 1857), Crassostrea corteziensis (Hertlein, 1951) and Crassostrea gigas (Thunberg, 1793) from the Mexican Pacific coast. Transportation of fresh oysters for human consumption and repopulation could be implicated in the transmission and dissemination of this parasite across the Mexican Pacific coast. The aim of this study was two-fold. First, we evaluated the P. marinus infection parameters by PCR and RFTM (Ray’s fluid thioglycollate medium) in C. virginica from four major lagoons (Términos Lagoon, Campeche; Carmen-Pajonal-Machona Lagoon complex, Tabasco; Mandinga Lagoon, Veracruz; and La Pesca Lagoon, Tamaulipas) from the Gulf of Mexico. Secondly, we used DNA sequence analyses of the ribosomal non-transcribed spacer (rNTS) region of P. marinus to determine the possible translocation of this species from the Gulf of Mexico to the Mexican Pacific coast.ResultsPerkinsus marinus prevalence by PCR was 57.7% (338 out of 586 oysters) and 38.2% (224 out of 586 oysters) by RFTM. The highest prevalence was observed in the Carmen-Pajonal-Machona Lagoon complex in the state of Tabasco (73% by PCR and 58% by RFTM) and the estimated weighted prevalence (WP) was less than 1.0 in the four lagoons. Ten unique rDNA-NTS sequences of P. marinus [termed herein the “P. marinus (Pm) haplotype”] were identified in the Gulf of Mexico sample. They shared 96–100% similarity with 18 rDNA-NTS sequences from the GenBank database which were derived from 16 Mexican Pacific coast infections and two sequences from the USA. The phylogenetic tree and the haplotype network showed that the P. marinus rDNA-NTS sequences from Mexico were distant from the rDNA-NTS sequences of P. marinus reported from the USA. The ten rDNA-NTS sequences described herein were restricted to specific locations displaying different geographical connections within the Gulf of Mexico; the Carmen-Pajonal-Machona Pm1 haplotype from the state of Tabasco shared a cluster with P. marinus isolates reported from the Mexican Pacific coast.ConclusionsThe rDNA-NTS sequences of P. marinus from the state of Tabasco shared high similarity with the reference rDNA-NTS sequences from the Mexican Pacific coast. The high similarity suggests a transfer of oysters infected with P. marinus from the Mexican part of the Gulf of Mexico into the Mexican Pacific coast.

The abundance of Fob1 modulates the efficiency of rRFBs to stall replication forks.

In eukaryotes, ribosomal genes (rDNA) are organized in tandem repeats localized in one or a few clusters. Each repeat encompasses a transcription unit and a non-transcribed spacer. Replication forks moving in the direction opposite to transcription are blocked at specific sites called replication fork barriers (rRFBs) in the non-transcribed spacer close to the 3′ end of the transcription unit. Here, we investigated and quantified the efficiency of rRFBs in Saccharomyces cerevisiae and to this end transfected budding yeast cells that express dissimilar quantities of Fob1 with circular minichromosomes containing different copies of the minimal 20-bp DNA segment that bind Fob1. To identify fork stalling we used high-resolution 2D agarose gel electrophoresis. The results obtained indicated that neighbor DNA sequences and the relative abundance of Fob1 modulate the efficiency of rRFBs to stall replication forks.

Lophopyrum Á. Löve (1980), Thinopyrum Á. Löve (1980), Trichopyrum Á. Löve (1986): one, two or three genera? A study based on the nuclear 5S DNA

Many species included in the genera Lophopyrum (A. Love 1980), Thinopyrum (A. Love 1980), and Trichopyrum (A. Love 1986) provide germplasm that can be used by breeders to improve commercial wheat cultivars through breeding programs. But various authors have classified them differently, placing them into one, two, three or even more genera. This lack of definition raises the question posed in the title; namely, one, two or three (or more) genera? To answer this question we created a data set of 480 5S nrDNA sequences from 15 species commonly assigned to one or more of these genera by different authors. Based upon the observed variation in DNA sequence of the 5S nrRNA gene mainly within the non-transcribed spacer region, sequences can be summarized into groups or classes of 5S nrDNA that we named unit classes. Unit classes can be assigned to different haplomes within the Triticeae tribe providing a means to study genome composition and species diversity among other ends. These results, where the species were defined on the basis of haplome composition, would then be used to test hypotheses that these species can be placed within a single genus or not. Contingent upon our analyses, we found that haplome composition does not follow the previously proposed taxonomies, based on cytogenetic data, and therefore that all 15 species investigated form one single genus.

High prevalence of superficial white onychomycosis by Trichophyton interdigitale in a Japanese nursing home with ageriatric hospital.

A mycological survey on feet was performed in a nursing home with a geriatric hospital to ascertain the prevalence of tinea lesions. Of 100 subjects, comprising 62 in the nursing home and 38 in the geriatric wing, 70 were diagnosed with tinea pedis, tinea unguium (onychomycosis) or both of which 54 had onychomycosis alone, nine tinea pedis alone and seven had co-existing onychomycosis and tinea pedis. The most common clinical type of onychomycosis was distal lateral subungual onychomycosis (DLSO) at 30 cases, followed by superficial white onychomycosis (SWO) at 23 cases. Fifteen strains of Trichophyton (T.) interdigitale isolated from 23 SWO patients comprised six molecular types (D2II, nine cases; C2II, two cases; four other types, one case of each), based on the non-transcribed spacer region (NTS) of the ribosomal DNA. The pathogen of three other SWO cases was identified as T.rubrum. Direct physical contact between the subjects was unlikely because they were bedridden most of the time. Nine T.interdigitale strains were isolated from a bathtub used by patients on the floor with a high incidence of SWO alone, and all nine strains were D2II type, which suggests nosocomial infection. Consequently, the hospital infection control policy committee was consulted, bathing arrangements were changed, and nursing staff were educated about onychomycosis.

A Tandemly Arranged Pattern of Two 5S rDNA Arrays in Amolops mantzorum (Anura, Ranidae)

In an attempt to extend the knowledge of the 5S rDNA organization in anurans, the 5S rDNA sequences of Amolops mantzorum were isolated, characterized, and mapped by FISH. Two forms of 5S rDNA, type I (209 bp) and type II (about 870 bp), were found in specimens investigated from various populations. Both of them contained a 118-bp coding sequence, readily differentiated by their non-transcribed spacer (NTS) sizes and compositions. Four probes (the 5S rDNA coding sequences, the type I NTS, the type II NTS, and the entire type II 5S rDNA sequences) were respectively labeled with TAMRA or digoxigenin to hybridize with mitotic chromosomes for samples of all localities. It turned out that all probes showed the same signals that appeared in every centromeric region and in the telomeric regions of chromosome 5, without differences within or between populations. Obviously, both type I and type II of the 5S rDNA arrays arranged in tandem, which was contrasting with other frogs or fishes recorded to date. More interestingly, all the probes detected centromeric regions in all karyotypes, suggesting the presence of a satellite DNA family derived from 5S rDNA.

Molecular breeding of Saccharomyces cerevisiae with high RNA content by harnessing essential ribosomal RNA transcription regulator

As yeast is commonly used for RNA production, it is industrially important to breed strains with high RNA contents. The upstream activating factor (UAF) plays an important role in transcription of ribosomal RNA (rRNA), a major constituent of intracellular RNA species. Here, we targeted the essential rRNA transcription regulator Rrn5 of Saccharomyces cerevisiae, a component of the UAF complex, and disrupted the genomic RRN5 gene using a helper plasmid carrying an RRN5 gene. Then we isolated nine suppressor mutants (Sup mutants) of RRN5 gene disruption, causing deficiency in rRNA transcription. The Sup mutants had RNA contents of approximately 40% of the wild type level and expansion of rDNA repeats to ca. 400–700 copies. Reintroduction of a functional RRN5 gene into Sup mutants caused a reduction in the number of rDNA repeats to close to the wild type level but did not change RNA content. However, we found that reintroduction of RRN5 into the Sup16 mutant (in which the FOB1 gene encoding the rDNA replication fork barrier site binding protein was disrupted) resulted in a significant increase (17%) in RNA content compared with wild type, although the rDNA repeat copy number was almost identical to the wild type strain. In this case, upregulated transcription of non-transcribed spacers (NTS) occurred, especially in the NTS2 region; this was likely mediated by RNA polymerase II and accounted for the increased RNA content. Thus, we propose a novel breeding strategy for developing high RNA content yeast by harnessing the essential rRNA transcription regulator.

Trypanosoma cruzi I genotype among isolates from patients with chronic Chagas disease followed at the Evandro Chagas National Institute of Infectious Diseases (FIOCRUZ, Brazil).

Trypanosoma cruzi is the etiologic agent of Chagas disease in humans, mainly in Latin America. Trypanosome stocks were isolated by hemoculture from patients followed at Evandro Chagas National Institute of Infectious Diseases (FIOCRUZ) and studied using different approaches. For species and genotype identification, the stocks were analyzed by parasitological techniques, polymerase chain reaction assays targeted to specific DNA sequences, isoenzyme patterns, besides sequencing of a polymorphic locus of TcSC5D gene (one stock). The isolates presented typical T. cruzi morphology and usually grew well in routine culture media. Metacyclic trypomastigotes were found in cultures or experimentally infected Triatoma infestans. All isolates were pure T. cruzi cultures, presenting typical 330-bp products from kinetoplast DNA minicircles, and 250 or 200-bp amplicons from the mini-exon non-transcribed spacer. Their genetic type assignment was resolved by their isoenzyme profiles. The finding of TcI in one asymptomatic patient from Paraíba was confirmed by the sequencing assay. TcVI was found in two asymptomatic individuals from Bahia and Rio Grande do Sul. TcII was identified in six patients from Pernambuco, Bahia and Minas Gerais, who presented different clinical forms: cardiac (2), digestive with megaesophagus (1), and indeterminate (3). The main T. cruzi genotypes found in Brazilian chronic patients were identified in this work, including TcI, which is less frequent and usually causes asymptomatic disease, unlike that in other American countries. This study emphasizes the importance of T. cruzi genotyping for possible correlations between the parasite and patient' responses to therapeutic treatment or disease clinical manifestations.

Molecular Strain Typing of Clinical Isolates, Trichophyton rubrum using Non Transcribed Spacer (NTS) Region as a Molecular Marker.

Dermatophytes are a group of fungi which infect keratinized tissues and causes superficial mycoses in humans and animals. The group comprises of three major genera, Trichophyton, Microsporum and Epidermophyton. Among them Trichophyton rubrum is a predominant anthropophilic fungi which causes chronic infections. Although, the infection is superficial and treatable, reinfection/coinfection causes inflation in the treatment cost. Identifying the source and mode of transmission is essential to prevent its transmission. Accurate discrimination is required to understand the clinical (relapse or reinfection) and epidemiological implications of the genetic heterogeneity of this species. Polymorphism in the Non Transcribed Spacer (NTS) region of ribosomal DNA (rDNA) clusters renders an effective way to discriminate strains among T. rubrum. To carry out the strain typing of the clinical isolates, Trichophyton rubrum using NTS as a molecular marker. Seventy T.rubrum clinical isolates obtained from April-2011-March 2013, from Sri Ramachandra Medical Centre, Chennai, Tamil Nadu, India, were identified by conventional phenotypic methods and included in this prospective study. The isolates were then subjected to Polymerase Chain Reaction (PCR) targeting two subrepeat elements (SREs), TRS-1 and TRS-2 of the NTS region. Strain-specific polymorphism was observed in both subrepeat loci. Total, nine different strains were obtained on combining both TRS-1 and TRS-2, SREs. The outcome has given a strong representation for using NTS region amplification in discriminating the T. rubrum clinical isolates. The method can be adapted as a tool for conducting epidemiology and population based study in T. rubrum infections. This will help in future exploration of the epidemiology of T. rubrum.

First report of Perkinsus honshuensis in the variegated carpet shell clam Ruditapes variegatus in Korea.

The recent discovery of Perkinsus honshuensis, a new Perkinsus species infecting Manila clams Ruditapes philippinarum (Sowerby, 1852), in Japan, suggested that, based on proximity, P. honshuensis could also be in Korean waters, where to date, P. olseni was believed to be the only Perkinsus species present. Perkinsus sp. infections consistently occurred among Ruditapes variegatus clams on a pebble beach on Jeju Island, off the south coast of Korea. The typical 'signet ring' morphology of the parasite was observed in the connective tissue of the digestive gland, and infection intensity was comparatively low (3.3 × 103 ± 1.2 × 104 to 1.3 × 104 ± 6.1 × 104 cells g-1 gill weight). Further DNA analyses of internal transcribed spacer (ITS-1, 5.8S and ITS-2) and non-transcribed spacer (NTS) regions of the parasite showed 98.9-99.8 and 98.5-99.5% similarity to those of P. honshuensis from Japan, respectively. Phylogenetic analyses using ITS and NTS sequences indicated that Perkinsus sp. from Jeju formed a highly supported clade with P. honshuensis. This is the first report of P. honshuensis infections in clams in Korean waters and the first report of R. variegatus as a host for that parasite.

Evidence of birth-and-death evolution of 5S rRNA gene in Channa species (Teleostei, Perciformes).

In higher eukaryotes, minor rDNA family codes for 5S rRNA that is arranged in tandem arrays and comprises of a highly conserved 120bp long coding sequence with a variable non-transcribed spacer (NTS). Initially the 5S rDNA repeats are considered to be evolved by the process of concerted evolution. But some recent reports, including teleost fishes suggested that evolution of 5S rDNA repeat does not fit into the concerted evolution model and evolution of 5S rDNA family may be explained by a birth-and-death evolution model. In order to study the mode of evolution of 5S rDNA repeats in Perciformes fish species, nucleotide sequence and molecular organization of five species of genus Channa were analyzed in the present study. Molecular analyses revealed several variants of 5S rDNA repeats (four types of NTS) and networks created by a neighbor net algorithm for each type of sequences (I, II, III and IV) did not show a clear clustering in species specific manner. The stable secondary structure is predicted and upstream and downstream conserved regulatory elements were characterized. Sequence analyses also shown the presence of two putative pseudogenes in Channa marulius. Present study supported that 5S rDNA repeats in genus Channa were evolved under the process of birth-and-death.

Molecular genetic features of 5S rDNA nontranscribed spacer in Hippophae rhamnoides L.

Amplification of sea buckthorn Hippophae rhamnoides L. 5S rDNA nontranscribed spacer with coding border anneal primers showed existence of a single fragment. The fragment was cloned and sequenced. It was shown that length of the Hippophae rhamnoides L. 5S rDNA nontranscribed spacer is 807 bp. Analysis of the sequence allowed to detect a high homology with early described microsatellite locuses of Hippophae rhamnoides L., russian olive Elaeagnus angustifolia L., and Calligonum mongolicum Turcz. that include a (GA)9 motif. These results may be useful to study a ribosomal RNA gene organization.

Molecular Markers Useful for Intraspecies Subtyping and Strain Differentiation of Dermatophytes.

Dermatophytosis is a very common skin disorder and the most frequent infection encountered by practicing dermatologists. The identification, pathogenicity, biology, and epidemiology of dermatophytes, the causative agents of dermatophytosis, are of interest for both dermatologists and medical mycologists. Recent advances in molecular methods have provided new techniques for identifying dermatophytes, including intraspecies variations. Intraspecies subtyping and strain differentiation have made possible the tracking of infections, the identification of common sources of infections, recurrence or reinfection after treatment, and analysis of strain virulence and drug resistance. This review describes molecular methods of intraspecies subtyping and strain differentiation, including analyses of mitochondrial DNA and non-transcribed spacer regions of ribosomal RNA genes, random amplification of polymorphic DNA, and microsatellite markers, along with their advantages and limitations.

A new set of rDNA-NTS-based multiple integrative cassettes for the development of antibiotic-marker-free recombinant yeasts

The traditional yeast Saccharomyces cerevisiae has been widely used as a host system to produce recombinant proteins and metabolites of great commercial value. To engineer recombinant yeast that stably maintains expression cassettes without an antibiotic resistance gene, we developed new multiple integration cassettes by exploiting the non-transcribed spacer (NTS) of ribosomal DNA (rDNA) in combination with defective selection markers. The 5′ and 3′-fragments of rDNA-NTS2 were used as flanking sequences for the expression cassettes carrying a set of URA3, LEU2, HIS3, and TRP1 selection markers with truncated promoters of different lengths. The integration numbers of NTS-based expression cassettes, ranging from one to ∼30 copies, showed a proportional increase with the extent of decreased expression of the auxotrophic markers. The NTS-based cassettes were used to construct yeast strains expressing the capsid protein of red-spotted grouper necrosis virus (RG-NNVCP) in a copy number-dependent manner. Oral administration of the recombinant yeast, harboring ∼30 copies of the integrated RG-NNVCP cassettes, provoked efficient immune responses in mice. In contrast, for the NTS cassettes expressing a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase, the integrant carrying only 4 copies was screened as the highest producer of squalene, showing a 150-fold increase compared to that of the wild-type strain. The multiple integrated cassettes were stably retained under prolonged nonselective conditions. Altogether, our results strongly support that rDNA-NTS integrative cassettes are useful tools to construct recombinant yeasts carrying optimal copies of a desired expression cassette without an antibiotic marker gene, which are suitable as oral vaccines or feed additives for animal and human consumption.

Evolutionary dynamics of satellite DNA repeats from Phaseolus beans.

Common bean (Phaseolus vulgaris) subtelomeres are highly enriched for khipu, the main satellite DNA identified so far in this genome. Here, we comparatively investigate khipu genomic organization in Phaseolus species from different clades. Additionally, we identified and characterized another satellite repeat, named jumper, associated to khipu. A mixture of P. vulgaris khipu clones hybridized in situ confirmed the presence of khipu-like sequences on subterminal chromosome regions in all Phaseolus species, with differences in the number and intensity of signals between species and when species-specific clones were used. Khipu is present as multimers of ∼500bp and sequence analyses of cloned fragments revealed close relationship among khipu repeats. The new repeat, named jumper, is a 170-bp satellite sequence present in all Phaseolus species and inserted into the nontranscribed spacer (NTS) of the 5S rDNA in the P. vulgaris genome. Nevertheless, jumper was found as a high-copy repeat at subtelomeres and/or pericentromeres in the Phaseolus microcarpus lineage only. Our data argue for khipu as an important subtelomeric satellite DNA in the genus and for a complex satellite repeat composition of P. microcarpus subtelomeres, which also contain jumper. Furthermore, the differential amplification of these repeats in subtelomeres or pericentromeres reinforces the presence of a dynamic satellite DNA library in Phaseolus.

Mechanism of Regulation of Intrachromatid Recombination and Long-Range Chromosome Interactions in Saccharomyces cerevisiae.

The NAD-dependent histone deacetylase Sir2 controls ribosomal DNA (rDNA) silencing by inhibiting recombination and RNA polymerase II-catalyzed transcription in the rDNA of Saccharomyces cerevisiae Sir2 is recruited to nontranscribed spacer 1 (NTS1) of the rDNA array by interaction between the RENT ( RE: gulation of N: ucleolar S: ilencing and T: elophase exit) complex and the replication terminator protein Fob1. The latter binds to its cognate sites, called replication termini (Ter) or replication fork barriers (RFB), that are located in each copy of NTS1. This work provides new mechanistic insights into the regulation of rDNA silencing and intrachromatid recombination by showing that Sir2 recruitment is stringently regulated by Fob1 phosphorylation at specific sites in its C-terminal domain (C-Fob1), which also regulates long-range Ter-Ter interactions. We show further that long-range Fob1-mediated Ter-Ter interactions in trans are downregulated by Sir2. These regulatory mechanisms control intrachromatid recombination and the replicative life span (RLS).