Evolutionary conservation of a functionally important backbone phosphate group critical for aminoacylation of histidine tRNAs

All histidine tRNA molecules have an extra nucleotide, G-1, at the 5' end of the acceptor stem. In bacteria, archaea, and eukaryotic organelles, G-1 base pairs with C73, while in eukaryotic cytoplasmic tRNAHis, G-1 is opposite A73. Previous studies of Escherichia coli histidyl-tRNA synthetase (HisRS) have demonstrated the importance of the G-1:C73 base pair to tRNAHis identity. Specifically, the 5'-monophosphate of G-1 and the major groove amine of C73 are recognized by E. coli HisRS; these individual atomic groups each contribute approximately 4 kcal/mol to transition state stabilization. In this study, two chemically synthesized 24-nucleotide RNA microhelices, each of which recapitulates the acceptor stem of either E. coli or Saccharomyces cervisiae tRNAHis, were used to facilitate an atomic group "mutagenesis" study of the -1:73 base pair recognition by S. cerevisiae HisRS. Compared with E. coli HisRS, microhelixHis is a much poorer substrate relative to full-length tRNAHis for the yeast enzyme. However, the data presented here suggest that, similar to the E. coli system, the 5' monophosphate of yeast tRNA(His) is critical for aminoacylation by yeast HisRS and contributes approximately 3 kcal/mol to transition state stability. The primary role of the unique -1:73 base pair of yeast tRNAHis appears to be to properly position the critical 5' monophosphate for interaction with the yeast enzyme. Our data also suggest that the eukaryotic HisRS/tRNAHis interaction has coevolved to rely less on specific major groove interactions with base atomic groups than the bacterial system.     

Portal-large terminase interactions of the bacteriophage T4 DNA packaging machine implicate a molecular lever mechanism for coupling ATPase to DNA translocation

DNA packaging by double-stranded DNA bacteriophages and herpesviruses is driven by a powerful molecular machine assembled at the portal vertex of the empty prohead. The phage T4 packaging machine consists of three components: dodecameric portal (gp20), pentameric large terminase motor (gp17), and 11- or 12-meric small terminase (gp16). These components dynamically interact and orchestrate a complex series of reactions to produce a DNA-filled head containing one viral genome per head. Here, we analyzed the interactions between the portal and motor proteins using a direct binding assay, mutagenesis, and structural analyses. Our results show that a portal binding site is located in the ATP hydrolysis-controlling subdomain II of gp17. Mutations at key residues of this site lead to temperature-sensitive or null phenotypes. A conserved helix-turn-helix (HLH) that is part of this site interacts with the portal. A recombinant HLH peptide competes with gp17 for portal binding and blocks DNA translocation. The helices apparently provide specificity to capture the cognate prohead, whereas the loop residues communicate the portal interaction to the ATPase center. These observations lead to a hypothesis in which a unique HLH-portal interaction in the symmetrically mismatched complex acts as a lever to position the arginine finger and trigger ATP hydrolysis. Transiently connecting the critical parts of the motor; subdomain I (ATP binding), subdomain II (controlling ATP hydrolysis), and C-domain (DNA movement), the portal-motor interactions might ensure tight coupling between ATP hydrolysis and DNA translocation.     

TEMPORAL VARIATION AT THE MHC CLASS IIB IN WILD POPULATIONS OF THE GUPPY (POECILIA RETICULATA)

Understanding genetic diversity in natural populations is a fundamental objective of evolutionary biology. The immune genes of the major histocompatibility complex (MHC) are excellent candidates to study such diversity because they are highly polymorphic in populations. Although balancing selection may be responsible for maintaining diversity at these functionally important loci, temporal variation in selection pressure has rarely been examined. We examine temporal variation in MHC class IIB diversity in nine guppy (Poecilia reticulata) populations over two years. We found that five of the populations changed significantly more at the MHC than at neutral (microsatellite) loci as measured by F_ST, which suggests that the change at the MHC was due to selection and not neutral processes. Additionally, pairwise population differentiation measures at the MHC were higher in 2007 than in 2006, with the signature of selection changing from homogenizing to diversifying selection or neutral evolution. Interestingly, within the populations the magnitude of the change at the MHC between years was related to the change in the proportion of individuals infected by a common parasite, indicating a link between genetic structure and the parasite. Our data thereby implicate temporal variation in selective pressure as an important mechanism maintaining diversity at the MHC in wild populations.     

Sleep and circadian rhythms in spousally bereaved seniors

A laboratory study of sleep and circadian rhythms was undertaken in 28 spousally bereaved seniors (> or =60 yrs) at least four months after the loss event. Measures taken included two nights of polysomnography (second night used), approximately 36 h of continuous core body temperature monitoring, and four assessments of mood and alertness throughout a day. Preceding the laboratory study, two-week diaries were completed, allowing the assessment of lifestyle regularity using the 17-item Social Rhythm Metric (SRM) and the timing of sleep using the Pittsburgh Sleep Diary (PghSD). Also completed were questionnaires assessing level of grief (Texas Revised Inventory of Grief [TRIG] and Index of Complicated Grief [ICG]), subjective sleep quality (Pittsburgh Sleep Quality Index [PSQI]), morningness-eveningness (Composite Scale of Morningness [CSM]), and clinical interview yielding a Hamilton Depression Rating Scale (HDRS) score. Grief was still present, as indicated by an average TRIG score of about 60. On average, the bereaved seniors habitually slept between approximately 23:00 and approximately 06:40 h, achieving approximately 6 h of sleep with a sleep efficiency of approximately 80%. They took about 30 min to fall asleep, and had their first REM episode after 75 min. About 20% of their sleep was in Stage REM, and about 3% in Stages 3 or 4 (slow wave sleep). Their mean PSQI score was 6.4. Their circadian temperature rhythms showed the usual classic shape with a trough at approximately 01:00 h, a fairly steep rise through the morning hours, and a more gradual rise to mid-evening, with an amplitude of approximately 0.8 degrees C. In terms of lifestyle regularity, the mean regularity (SRM) score was 3.65 (slightly lower than that usually seen in seniors). Mood and alertness showed time-of-day variation with peak alertness in the late morning and peak mood in the afternoon. Correlations between outcome sleep/circadian variables and level of grief (TRIG score) were calculated; there was a slight trend for higher grief to be associated with less time spent asleep (p=0.07) and reduced alertness at 20:00 h (p=0.05). Depression score was not correlated with TRIG score (p>0.20). When subjects were divided into groups by the nature of their late spouse's death (expected/after a long-term chronic illness [n=18] versus unexpected [n=10]), no differences emerged in any of the variables. In conclusion, when studied at least four months after the loss event, there appears to be some sleep disruption in spousally bereaved seniors. However, this disruption does not appear to be due to bereavement-related disruptions in the circadian system.     

iWRAP: An interface threading approach with application to prediction of cancer-related protein-protein interactions

Current homology modeling methods for predicting protein-protein interactions (PPIs) have difficulty in the “twilight zone” (< 40%) of sequence identities. Threading methods extend coverage further into the twilight zone by aligning primary sequences for a pair of proteins to a best-fit template complex to predict an entire three-dimensional structure. We introduce a threading approach, iWRAP, which focuses only on the protein interface. Our approach combines a novel linear programming formulation for interface alignment with a boosting classifier for interaction prediction. We demonstrate its efficacy on SCOPPI, a classification of PPIs in the Protein Databank, and on the entire yeast genome. iWRAP provides significantly improved prediction of PPIs and their interfaces in stringent cross-validation on SCOPPI. Furthermore, by combining our predictions with a full-complex threader, we achieve a coverage of 13% for the yeast PPIs, which is close to a 50% increase over previous methods at a higher sensitivity. As an application, we effectively combine iWRAP with genomic data to identify novel cancer-related genes involved in chromatin remodeling, nucleosome organization, and ribonuclear complex assembly. iWRAP is available at http://iwrap.csail.mit.edu.     

A Study of the Vaginal Microbiome in Healthy Canadian Women Utilizing cpn60-Based Molecular Profiling Reveals Distinct Gardnerella Subgroup Community State Types

The vaginal microbiota is important in women’s reproductive and overall health. However, the relationships between the structure, function and dynamics of this complex microbial community and health outcomes remain elusive. The objective of this study was to determine the phylogenetic range and abundance of prokaryotes in the vaginal microbiota of healthy, non-pregnant, ethnically diverse, reproductive-aged Canadian women. Socio-demographic, behavioural and clinical data were collected and vaginal swabs were analyzed from 310 women. Detailed profiles of their vaginal microbiomes were generated by pyrosequencing of the chaperonin-60 universal target. Six community state types (CST) were delineated by hierarchical clustering, including three Lactobacillus-dominated CST (L. crispatus, L. iners, L. jensenii), two Gardnerella-dominated (subgroups A and C) and an “intermediate” CST which included a small number of women with microbiomes dominated by seven other species or with no dominant species but minority populations of Streptococcus, Staphylococcus, Peptoniphilus, E. coli and various Proteobacteria in co-dominant communities. The striking correspondence between Nugent score and deep sequencing CST continues to reinforce the basic premise provided by the simpler Gram stain method, while additional analyses reveal detailed cpn60-based phylogeny and estimated abundance in microbial communities from vaginal samples. Ethnicity was the only demographic or clinical characteristic predicting CST, with differences in Asian and White women (p = 0.05). In conclusion, this study confirms previous work describing four cpn60-based subgroups of Gardnerella, revealing previously undescribed CST. The data describe the range of bacterial communities seen in Canadian women presenting with no specific vaginal health concerns, and provides an important baseline for future investigations of clinically important cohorts.     

The Novel Application of Non-Lethal Citizen Science Tissue Sampling in Recreational Fisheries

Increasing fishing pressure and uncertainty surrounding recreational fishing catch and effort data promoted the development of alternative methods for conducting fisheries research. A pilot investigation was undertaken to engage the Australian game fishing community and promote the non-lethal collection of tissue samples from the black marlin Istiompax indica, a valuable recreational-only species in Australian waters, for the purpose of future genetic research. Recruitment of recreational anglers was achieved by publicizing the project in magazines, local newspapers, social media, blogs, websites and direct communication workshops at game fishing tournaments. The Game Fishing Association of Australia and the Queensland Game Fishing Association were also engaged to advertise the project and recruit participants with a focus on those anglers already involved in the tag-and-release of marlin. Participants of the program took small tissue samples using non-lethal methods which were stored for future genetic analysis. The program resulted in 165 samples from 49 participants across the known distribution of I. indica within Australian waters which was a sufficient number to facilitate a downstream population genetic analysis. The project demonstrated the potential for the development of citizen science sampling programs to collect tissue samples using non-lethal methods in order to achieve targeted research objects in recreationally caught species.     

Ethylene directs auxin to control root cell expansion

Root morphogenesis is controlled by the regulation of cell division and expansion. We isolated an allele of the eto1 ethylene overproducer as a suppressor of the auxin-resistant mutant ibr5, prompting an examination of crosstalk between the phytohormones auxin and ethylene in control of root epidermal cell elongation and root hair elongation. We examined the interaction of eto1 with mutants that have reduced auxin response or transport and found that ethylene overproduction partially restored auxin responsiveness to these mutants. In addition, we found that the effects of endogenous ethylene on root cell expansion in eto1 seedlings were partially impeded by dampening auxin signaling, and were fully suppressed by blocking auxin influx. These data provide insight into the interaction between these two key plant hormones, and suggest that endogenous ethylene directs auxin to control root cell expansion.     

Leveraging genomic resources of model species for the assessment of diversity and phylogeny in wild and domesticated lentil

Advances in comparative genomics have provided significant opportunities for analysis of genetic diversity in species with limited genomic resources, such as the genus Lens. Medicago truncatula expressed sequence tags (ESTs) were aligned with the Arabidopsis thaliana genome sequence to identify conserved exon sequences and splice sites in the ESTs. Conserved primers (CPs) based on M. truncatula EST sequences flanking one or more introns were then designed. A total of 22% of the CPs produced polymerase chain reaction amplicons in lentil and were used to sequence amplicons in 175 wild and 133 domesticated lentil accessions. Analysis of the sequences confirmed that L. nigricans and L. ervoides are well-defined species at the DNA sequence level. Lens culinaris subsp. odemensis, L. culinaris subsp. tomentosus, and L. lamottei may constitute a single taxon pending verification with crossability experiments. Lens culinaris subsp. orientalis is the progenitor of domesticated lentil, L. culinaris subsp. culinaris (as proposed before), but a more specific area of origin can be suggested in southern Turkey. We were also able to detect the divergence, following domestication, of the domesticated gene pool into overlapping large-seeded (megasperma) and small-seeded (microsperma) groups. Lentil domestication led to a loss of genetic diversity of approximately 40%. The approach followed in this research has allowed us to rapidly exploit sequence information from model plant species for the study of genetic diversity of a crop such as lentil with limited genomic resources.