Cassandra retrotransposons carry independently transcribed 5S RNA.

We report a group of TRIMs (terminal-repeat retrotransposons in miniature), which are small nonautonomous retrotransposons. These elements, named Cassandra, universally carry conserved 5S RNA sequences and associated RNA polymerase (pol) III promoters and terminators in their long terminal repeats (LTRs). They were found in all vascular plants investigated. Uniquely for LTR retrotransposons, Cassandra produces noncapped, polyadenylated transcripts from the 5S pol III promoter. Capped, read-through transcripts containing Cassandra sequences can also be detected in RNA and in EST databases. The predicted Cassandra RNA 5S secondary structures resemble those for cellular 5S rRNA, with high information content specifically in the pol III promoter region. Genic integration sites are common for Cassandra, an unusual feature for abundant retrotransposons. The 5S in each LTR produces a tandem 5S arrangement with an inter-5S spacing resembling that of cellular 5S. The distribution of 5S genes is very variable in flowering plants and may be partially explained by Cassandra activity. Cassandra thus appears both to have adapted a ubiquitous cellular gene for ribosomal RNA for use as a promoter and to parasitize an as-yet-unidentified group of retrotransposons for the proteins needed in its lifecycle.

Bacterial intein-like domains of predatory bacteria: a new domain type characterized in Bdellovibrio bacteriovorus.

We report a new family of bacterial intein-like domains (BILs) identified in ten proteins of four diverse predatory bacteria. BILs belong to the HINT (Hedgehog/Intein) superfamily of domains that post-translationally self-process their protein molecules by protein splicing and self-cleavage. The new, C-type, BILs appear with other domains, including putative predator-specific domain 1 (PPS-1), a new domain typically appearing immediately upstream of C-type BILs. The Bd2400 protein of the obligate predator Bdellovibrio bacteriovorus includes a C-type BIL and a PPS-1 domains at its C-terminal part, and a signal peptide and two polycystic kidney disease domains at its N-terminal part. We demonstrate the in vivo transcription, translation, secretion, and processing of the B. bacteriovorus protein, and the in vitro autocatalytic N-terminal cleavage activity of its C-type BIL. Interestingly, whereas the Bd2400 gene is constitutively expressed, its protein product is differentially processed throughout the dimorphic life cycle of the B. bacteriovorus predator. The modular structure of the protein, its localization, and complex processing suggest that it may be involved in the interaction between the predator and its prey.

Use of chimeric DNA-RNA primers in quantitative PCR for detection of Ehrlichia canis and Babesia canis.

To overcome the problem of nonspecific by-products in quantitative PCR (qPCR) assays, we constructed DNA-RNA chimeric primers and evaluated their use in the detection and quantification of the Ehrlichia canis 16S rRNA, Babesia canis Hsp70, and canine beta-actin genes. Several RNA bases were incorporated into specific positions in the DNA primers, while no RNA stretches were allowed. qPCR reactions were carried out without preamplification steps. This resulted in decreased formation of undesirable by-products and a 10-fold increase in assay sensitivity.

HCV genotype-1 subtypes and resistance-associated substitutions in drug-naive and in direct-acting antiviral treatment failure patients.

BACKGROUND: Direct-acting antiviral (DAA) treatment regimens and response rates of patients with HCV genotype-1 (GT1) are currently considered subtype-dependent. Identification of clinically relevant resistance-associated substitutions (RASs) in the NS3 and NS5A proteins at baseline and in DAA failures, may also impact clinical decisions. METHODS: In a multicentre cohort study (n=308), NS3 or NS5B sequencing (n=248) was used to discriminate between GT1 subtypes. The correlation between baseline NS3 and NS5A RASs on the 12-week sustained virological response (SVR12) rates of 160 of the patients treated with second-generation DAAs was also assessed. Post-treatment resistance analysis was performed on samples from 58 patients exhibiting DAA virological failure. RESULTS: GT1a, GT1b and GT1d subtypes were identified in 23.0%, 75.4% and 1.2% of tested samples. GT1b was most prevalent (97.7%, 128/131) among patients born in the former Soviet Union. The Q80K NS3 RAS was identified in 17.5% (10/57) of the GT1a carriers, most of whom were Israeli-born. NS3 and NS5A baseline RASs showed a negligible correlation with SVR12 rates. Treatment-emergent RASs were observed among 8.9% (4/45) and 76.9% (10/13) of first- and second-generation DAA failures, respectively, with D168V/E (NS3), Y93H and L31M (NS5A) being the most prevalent mutations. CONCLUSIONS: NS3 sequencing analysis can successfully discriminate between GT1 subtypes and identify NS3 amino acid substitutions. While pre-treatment NS3 and NS5A RASs marginally affect second-generation DAA SVR12 rates, post-treatment resistance analysis should be considered prior to re-therapy.

Hidden messages in the nef gene of human immunodeficiency virus type 1 suggest a novel RNA secondary structure.

The coexistence of multiple codes in the genome of human immunodeficiency virus type 1 (HIV-1) was analyzed. We explored factors constraining the variability of the virus genome primarily in relation to conserved RNA secondary structures overlapping coding sequences, and used a simple combination of algorithms for RNA secondary structure prediction based on the nearest-neighbor thermodynamic rules and a statistical approach. In our previous study, we applied this combination to a non- redundant data set of env nucleotide sequences, confirmed the conservative secondary structure of the rev-responsive element (RRE) and found a new RNA structure in the first conserved (C1) region of the env gene. In this study, we analyzed the variability of putative RNA secondary structures inside the nef gene of HIV-1 by applying these algorithms to a non-redundant data set of 104 nef sequences retrieved from the Los Alamos HIV database, and predicted the existence of a novel functional RNA secondary structure in the beta3/beta4 regions of nef. The predicted RNA fold in the beta3/beta4 region of nef appears in two forms with different loop sizes. The loop of the first fold consists of seven nucleotides (positions 494-500), with consensus UCAAGCU appearing in 79% of sequences. The other has a five-base loop (positions 495-499) with consensus CAAGC. The difference in size between these two loops may reflect the difference between respective counterparts in the hairpin recognition. This may also have an adaptive biological significance.

Large retrotransposon derivatives: abundant, conserved but nonautonomous retroelements of barley and related genomes.

Retroviruses and LTR retrotransposons comprise two long-terminal repeats (LTRs) bounding a central domain that encodes the products needed for reverse transcription, packaging, and integration into the genome. We describe a group of retrotransposons in 13 species and four genera of the grass tribe Triticeae, including barley, with long, approximately 4.4-kb LTRs formerly called Sukkula elements. The approximately 3.5-kb central domains include reverse transcriptase priming sites and are conserved in sequence but contain no open reading frames encoding typical retrotransposon proteins. However, they specify well-conserved RNA secondary structures. These features describe a novel group of elements, called LARDs or large retrotransposon derivatives (LARDs). These appear to be members of the gypsy class of LTR retrotransposons. Although apparently nonautonomous, LARDs appear to be transcribed and can be recombinationally mapped due to the polymorphism of their insertion sites. They are dispersed throughout the genome in an estimated 1.3 x 10(3) full-length copies and 1.16 x 10(4) solo LTRs, indicating frequent recombinational loss of internal domains as demonstrated also for the BARE-1 barley retrotransposon.

RNA secondary structure and squence conservation in C1 region of human immunodeficiency virus type 1 env gene.

We have analyzed amino acid, nucleotide sequence, and RNA secondary structure variability in the env gene of human immunodeficiency virus type (HIV-1). In applying algorithms for computing optimal RNA-folding patterns to a nonredundant data set of 178 env nucleotide sequences, we found a conserved RNA stem-loop structure in the first conserved (C1) region of the env gene. This detailed examination also revealed the known secondary structure conservation of the Rev-responsive element (RRE). This finding is also supported by a higher third position conservation of the translatable reading frame along these subregions. The typical folding of the C1 region consists of two isolated stem-loop structures. These highly conserved structures are likely to have a biological function. This assumption is supported by the conservation of the third position along the coding region of these structures. The third position retains a conservation level above what would be statistically expected.

Multiplex real-time qPCR for the detection of Ehrlichia canis and Babesia canis vogeli.

Ehrlichia canis and Babesia canis vogeli are two tick-borne canine pathogens with a worldwide importance. Both pathogens are transmitted by Rhipicephalus sanguineus, the brown dog tick, which has an increasing global distribution. A multiplex quantitative real-time PCR (qPCR) assay for the simultaneous detection of the tick-borne pathogens E. canis and B. canis vogeli was developed using dual-labeled probes. The target genes were the 16S rRNA of E. canis and the heat shock protein 70 (hsp70) of B. canis vogeli. The canine beta actin (ACTB) gene was used as an internal control gene. The assay was conducted without using any pre-amplification steps such as nested reactions. The sensitivity of each reaction in the multiplex qPCR assay was tested in the presence of high template concentrations of the other amplified genes in the same tube and in the presence of canine DNA. The detection threshold of the multiplex assay was 1-10 copies/µl in all channels and the amplifications of the B. canis hsp70 and ACTB were not affected by the other simultaneous reactions, while minor interference was observed in the amplification of the E. canis 16S rRNA gene. This assay would be useful for diagnostic laboratories and may save time, labor and costs.

Leishmania tropica in rock hyraxes (Procavia capensis) in a focus of human cutaneous leishmaniasis.

Cutaneous leishmaniasis, caused by Leishmania tropica, has recently emerged in urban and rural foci of central and northern Israel, and constitutes a major public health concern. Rock hyraxes (Procavia capensis), the suspected natural reservoir, were trapped in the cutaneous leishmaniasis urban focus of Maale Adumim in central Israel and evaluated for L. tropica infection by real-time kinetoplast DNA (kDNA) polymerase chain reaction (PCR) and serology. Real-time PCR on blood and computerized western blot serology analysis was positive for L. tropica in 58% and 80%, respectively, of the hyraxes tested. Phylogenetic analysis of the ribosomal internal transcribed spacer 1 region indicated that similar genotypes were present in humans and hyraxes from the same habitat. The high rates of infection and exposure to L. tropica among hyraxes supports their involvement in the transmission cycle of this parasite, and their potential role as a reservoir for human disease.

Hereditary family signature of facial expression.

Although facial expressions of emotion are universal, individual differences create a facial expression "signature" for each person; but, is there a unique family facial expression signature? Only a few family studies on the heredity of facial expressions have been performed, none of which compared the gestalt of movements in various emotional states; they compared only a few movements in one or two emotional states. No studies, to our knowledge, have compared movements of congenitally blind subjects with their relatives to our knowledge. Using two types of analyses, we show a correlation between movements of congenitally blind subjects with those of their relatives in think-concentrate, sadness, anger, disgust, joy, and surprise and provide evidence for a unique family facial expression signature. In the analysis "in-out family test," a particular movement was compared each time across subjects. Results show that the frequency of occurrence of a movement of a congenitally blind subject in his family is significantly higher than that outside of his family in think-concentrate, sadness, and anger. In the analysis "the classification test," in which congenitally blind subjects were classified to their families according to the gestalt of movements, results show 80% correct classification over the entire interview and 75% in anger. Analysis of the movements' frequencies in anger revealed a correlation between the movements' frequencies of congenitally blind individuals and those of their relatives. This study anticipates discovering genes that influence facial expressions, understanding their evolutionary significance, and elucidating repair mechanisms for syndromes lacking facial expression, such as autism.

Overlapping messages and survivability.

The phenomenon of overlapping of various sequence messages in genomes is a puzzle for evolutionary theoreticians, geneticists, and sequence researchers. The overlapping is possible due to degeneracy of the messages, in particular, degeneracy of codons. It is often observed in organisms with a limited size of genome, possessing polymerases of low fidelity. The most accepted view considers the overlapping as a mechanism to increase the amount of information per unit length. Here we present a model that suggests direct evolutionary advantage of the message overlapping. Two opposing drives are considered: (a) reduction in the amount of vulnerable points when the overlapping of two messages involves common critical points and (b) cumulative compromising cost of coexistence of messages at the same site. Over a broad range of conditions the reduction of the target size prevails, thus making the overlapping of messages advantageous.