Microsatellites in different eukaryotic genomes: survey and analysis

We examined the abundance of microsatellites with repeated unit lengths of 1-6 base pairs in several eukaryotic taxonomic groups: primates, rodents, other mammals, nonmammalian vertebrates, arthropods, Caenorhabditis elegans, plants, yeast, and other fungi. Distribution of simple sequence repeats was compared between exons, introns, and intergenic regions. Tri- and hexanucleotide repeats prevail in protein-coding exons of all taxa, whereas the dependence of repeat abundance on the length of the repeated unit shows a very different pattern as well as taxon-specific variation in intergenic regions and introns. Although it is known that coding and noncoding regions differ significantly in their microsatellite distribution, in addition we could demonstrate characteristic differences between intergenic regions and introns. We observed striking relative abundance of (CCG)(n)*(CGG)(n) trinucleotide repeats in intergenic regions of all vertebrates, in contrast to the almost complete lack of this motif from introns. Taxon-specific variation could also be detected in the frequency distributions of simple sequence motifs. Our results suggest that strand-slippage theories alone are insufficient to explain microsatellite distribution in the genome as a whole. Other possible factors contributing to the observed divergence are discussed.     

Behavioural responses to perceived risk of tick-borne encephalitis: vaccination and avoidance in the Baltics and Slovenia

Tick-borne encephalitis (TBE) incidence increased markedly in the Baltics and Slovenia in the early 1990s, but then declined again in some places. Our analyses of temporal and spatial data on TBE incidence and vaccination revealed that over 1970-2005 up-take of vaccination varied in both time and space according to incidence, i.e. was apparently responsive to perceived risk. Since 1999, however, decreases in incidence in many counties within each country have far exceeded vaccination rates or immunity through natural exposure, and in Latvia and Lithuania these changes are correlated with previous incidence. Survey data on human activities in Latvia revealed that people in socio-economic groups whose behaviour put them at highest risk of exposure to ticks in forests, including people with lower education and lowest incomes, are least likely to be vaccinated. We conclude that risk avoidance through changing human behaviour has driven incidence-dependent decreases in TBE infection, but targeted vaccination campaigns could provide more secure protection.     

Enhanced malignant transformation induced by expression of a distinct protein domain of ribonucleotide reductase large subunit from herpes simplex virus type 2.

The 1.3-kilobase (kb) Pst I DNA fragment C (Pst I-C) of herpes simplex virus type 2 (HSV-2) morphological transforming region III (mtrIII; map unit 0.562-0.570) encodes part of the N-terminal half of the large subunit of ribonucleotide reductase (RR1; amino acid residues 71-502) and induces the neoplastic transformation of immortalized cell lines. To assess directly the role of these RR1 protein sequences in cell transformation, the Pst I-C fragment was cloned in an expression vector (p91023) containing an adenovirus-simian virus 40 promoter-enhancer to generate recombinant plasmid p9-C. Expression of a protein domain (approximately 65 kDa) was observed in p9-C-transfected COS-7 and Rat2 cells but not in those transfected with plasmid pHC-14 (Pst I-C in a promoterless vector). In Rat2 cells, p9-C induced highly transformed foci at an elevated frequency compared with that of pHC-14. Introduction of translation termination (TAG) condons within the RR1 coding sequence and within all three reading frames inactivated RR1 protein expression from p9-C and reduced its transforming activity to the level seen with the standard pHC-14 construct. Wild-type p9-C specified a protein kinase capable of autophosphorylation. Computer-assisted analysis further revealed significant similarity between regions of mtrIII-specific RR1 and amino acid patterns conserved within the proinsulin precursor family and DNA transposition proteins. These results identify a distinct domain of the HSV-2 RR1 protein involved in the induction of enhanced malignant transformation. In addition, the data indicate that the mtrIII DNA itself can induce basal-level transformation in the absence of protein expression.     

Molecular recognition of human ephrinB2 cell surface receptor by an emergent African henipavirus

The discovery of African henipaviruses (HNVs) related to pathogenic Hendra virus (HeV) and Nipah virus (NiV) from Southeast Asia and Australia presents an open-ended health risk. Cell receptor use by emerging African HNVs at the stage of host-cell entry is a key parameter when considering the potential for spillover and infection of human populations. The attachment glycoprotein from a Ghanaian bat isolate (GhV-G) exhibits <30% sequence identity with Asiatic NiV-G/HeV-G. Here, through functional and structural analysis of GhV-G, we show how this African HNV targets the same human cell-surface receptor (ephrinB2) as the Asiatic HNVs. We first characterized this virus−receptor interaction crystallographically. Compared with extant HNV-G–ephrinB2 structures, there was significant structural variation in the six-bladed β-propeller scaffold of the GhV-G receptor-binding domain, but not the Greek key fold of the bound ephrinB2. Analysis revealed a surprisingly conserved mode of ephrinB2 interaction that reflects an ongoing evolutionary constraint among geographically distal and phylogenetically divergent HNVs to maintain the functionality of ephrinB2 recognition during virus–host entry. Interestingly, unlike NiV-G/HeV-G, we could not detect binding of GhV-G to ephrinB3. Comparative structure–function analysis further revealed several distinguishing features of HNV-G function: a secondary ephrinB2 interaction site that contributes to more efficient ephrinB2-mediated entry in NiV-G relative to GhV-G and cognate residues at the very C terminus of GhV-G (absent in Asiatic HNV-Gs) that are vital for efficient receptor-induced fusion, but not receptor binding per se. These data provide molecular-level details for evaluating the likelihood of African HNVs to spill over into human populations.The emergence of negative-sense, single-stranded RNA viruses belonging to the genus Henipavirus, family Paramyxoviridae, epitomizes the increasing threat of zoonotic viruses to human health (1). Since the discovery of the highly pathogenic Nipah virus (NiV) and Hendra virus (HeV) in the 1990s, >20 henipaviruses (HNVs) have been detected throughout Africa, Asia, Australia, and Central America (2, 3). NiV is the prototypic member of this group and enzoonitically resides (>50% seroprevalence in some instances) in Old World fruit bat populations throughout Australasia (4). Zoonotic transfer of NiV to human populations from these natural reservoirs (5), sometimes through an animal intermediary such as pigs (6), leads to rapid-onset encephalitis with case-fatality rates >90% (1). Following zoonosis, some cases of person-to-person transmission have been observed (7, 8). Although bats are the predominant host reservoir, a putative henipa-like virus (HNLV), Mojiang virus (MojV), associated with severe pneumonia and three case fatalities, has also been isolated from rats in China (9). The extreme pathogenicity and potential for misuse has led to the designation of NiV and HeV as high-priority agents that require handling under biosafety-level-four conditions.The detection of 19 distinct clades of HNV in Africa (2, 10, 11) correlates with the broad geographic distribution and wide-ranging migrational patterns of the fruit bat host reservoir, Eidolon helvum (11). The remarkably high seroprevalence (∼40%) of HNV cross-reactive antibodies and the localization of many E. helvum communities near African towns and cities underscores the potential risk of spillover events into human populations (11, 12). Indeed, NiV cross-neutralizing antibodies have been detected in the sera of humans living in Cameroon (12). That these antibodies were found exclusively in individuals at high risk for zoonotic transmission, such as those that slaughter bats for bushmeat consumption and sale, suggests that such spillover events can occur. Whether or not African HNVs are as pathogenic to humans as NiV or HeV remains to be determined. Although it has also been suggested that these viruses may be the causative agent of misdiagnosed encephalitis-associated malaria (2, 13, 14), it is likely that the divergent clades of African HNVs are also diverse in their pathogenic potential.HNV entry into a host cell is a pH-independent process orchestrated by two membrane-anchored glycoproteins, HNV-G and -F (15). These viral glycoproteins interdependently facilitate cellular attachment and fusion, whereby receptor recognition by HNV-G at the cell surface triggers rearrangements in the HNV-F fusion glycoprotein (16). HNV-G is an oligomeric membrane protein, consisting of a short N-terminal cytoplasmic tail, a transmembrane region, an oligomerization-inducing stalk region, and a receptor-binding C-terminal six-bladed β-propeller. Identification of the ubiquitously expressed cell-surface signaling glycoproteins, ephrinB2 and ephrinB3, as functional receptors used during viral attachment by NiV and HeV has been key to understanding the broad tissue tropism of these viruses (16–21). Structural investigations of these ephrins in complex with NiV- and HeV-G have revealed the molecular determinants for host-cell recognition and zoonosis (22–26).In contrast to the wealth of available NiV and HeV genome sequences, only one African HNV has been sequenced to entirety, but it has not yet been isolated (2, 27). The sequence of this putative HNV (Gh-M74a; termed here as GhV) was derived from a bat in Ghana and is genetically distinct from Asiatic HeV and NiV (2). In contrast to NiV- and HeV-G, which are genetically quite similar (80% sequence identity), the putative GhV attachment glycoprotein from this virus, GhV-G, exhibits very limited sequence identity (<30%) with its Asiatic counterparts. Despite this genetic distance, ephrinB2 has been suggested as a functional interaction partner for this virus (27, 28). The conserved use of this receptor by GhV-G and Asiatic HNVs supports a general mechanism for HNV zoonosis in human populations. The likelihood of zoonotic transmission and the pathogenicity of such zoonotic viruses may depend, at least in part, on what adaptations are necessary for efficient use of the host receptor(s).Here, we determined the molecular basis for the interaction between GhV-G and ephrinB2 by X-ray crystallographic analysis. Despite the varied architecture of the henipaviral β-propeller scaffold between GhV-G and Asiatic HNV-Gs, we observed a highly conserved mode of ephrinB2 engagement. However, we also identify a secondary ephrinB2 interaction site that contributes to the more efficient receptor-mediated entry exhibited by NiV-G relative to GhV-G. These data verify a conserved HNV cell-attachment strategy for African and pathogenic Asiatic HNVs and establish a mechanism by which humans may be susceptible to African HNV infection.     

The influence of reading direction on hemianopic reading disorders

Hemianopic reading impairment is a consequence of a visual field defect to either the right or the left side and is characterized by an increased reading time and reduced reading performance. Depending on the side of the visual field defect, reading will be affected differently: Patients suffering from a visual field defect to the right side have noticeable difficulties in reading fluently with slowing. Patients suffering from a visual field defect to the left usually struggle to find the beginning of a line and read more fluently. It was suggested in the literature that changing the reading direction from horizontal to vertical may be a training strategy to reduce reading problems in patients with hemianopia. The aim of the study was to investigate the influence of reading direction on reading speed in patients with left- or right-sided visual field defects and in healthy controls. Method: In 13 patients with hemianopia and in 13 age-matched controls, reading speed was calculated for texts in standard as well as in clockwise rotated orientation of 90, 180, and 270°. Results: In both groups, text rotation reduced reading speed compared to standard reading. Patients with left-sided hemianopia had the greatest reduction after text rotation. Patients with right-sided hemianopia had the smallest speed reduction in 90° vertically rotated texts. Conclusions: Text rotation has different effects in left- or right-sided hemianopia patients. For patients with left-sided heminanopia, rotation of the text may not be a helpful training strategy, for right-sided hemianopia vertical rotation of the text of 90° may be a beneficial training strategy to reduce reading deficits.     

Evolution of the tumor suppressor BRCA1 locus in primates: implications for cancer predisposition

Germ-line mutations in the BRCA1 gene predispose affected individuals to breast and ovarian cancer syndromes. In an attempt to systematically analyze a broader spectrum of genetic changes ranging from frequent exon deletions and duplications to amino acid replacements and protein truncations, we isolated and characterized full size BRCA1 homologues from a representative group of non-human primates. Our analysis represents the first comprehensive sequence comparison of primate BRCA1 loci and corresponding proteins. The comparison revealed an unusually high proportion of indels in non-coding DNA. The major force driving evolutionary changes in non-coding BRCA1 sequences was Alu-mediated rearrangements, including Alu transpositions and Alu-associated deletions, indicating that structural instability of this locus may be intrinsic in anthropoids. Analysis of the non-synonymous/synonymous ratio in coding portions of the gene revealed the presence of both conserved and rapidly evolving regions in the BRCA1 protein. Previously, a rapidly evolving region with evidence of positive evolutionary selection in human and chimpanzee had been identified only in exon 11. Here, we show that most of the internal BRCA1 sequence is variable between primates and evolved under positive selection. In contrast, the terminal regions of BRCA1, which encode the RING finger and BRCT domains, experienced negative selection, which left them almost identical between the compared primates. Distribution of the reported missense mutations, but not frameshift and nonsense mutations, is positively correlated with BRCA1 protein conservation. Finally, on the basis of protein sequence conservation, we identified missense changes that are likely to compromise BRCA1 function.     

Identification of three clinically relevant Borrelia burgdorferi sensu lato genospecies by PCR-restriction fragment length polymorphism analysis of 16S-23S ribosomal DNA spacer amplicons

We report the results of a study of the prevalences of three clinically relevant Borrelia burgdorferi sensu lato genospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii) in 1,040 questing Ixodes ticks from all regions of Latvia, where Lyme borreliosis is endemic. The prevalences of Borrelia in Ixodes ricinus and Ixodes persulcatus were 22.6 and 27.9%, respectively. Molecular typing of B. burgdorferi from infected ticks was performed by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified fragments of the 16S-23S (rrs-rrlA) rRNA intergenic spacer by using species-specific primers and subsequent sequencing. The dominant Borrelia species in both Ixodes species was B. afzelii. In addition, different restriction patterns of B. garinii and B. afzelii were also identified. This study demonstrates that the 16S-23S rRNA PCR-RFLP typing method is simple, sensitive, and fast and that it allows one to differentiate among B. burgdorferi species and subspecies with various degrees of pathogenic potential directly in ticks. These features are important in monitoring Lyme disease.