Mutually exclusive RNA secondary structures regulate translation initiation of DinQ in Escherichia coli.

Protein translation can be affected by changes in the secondary structure of mRNA. The dinQ gene in Escherichia coli encodes a primary transcript (+1) that is inert to translation. Ribonucleolytic removal of the 44 first nucleotides converts the +1 transcript into a translationally active form, but the mechanism behind this structural change is unknown. Here we present experimental evidence for a mechanism where alternative RNA secondary structures in the two dinQ mRNA variants affect translation initiation by mediating opening or closing of the ribosome binding sequence. This structural switch is determined by alternative interactions of four sequence elements within the dinQ mRNA and also by the agrB antisense RNA. Additionally, the structural conformation of +1 dinQ suggests a locking mechanism comprised of an RNA stem that both stabilizes and prevents translation initiation from the full-length dinQ transcript. BLAST search and multiple sequence alignments define a new family of dinQ-like genes widespread in Enterobacteriaceae with close RNA sequence similarities in their 5' untranslated regions. Thus, it appears that a whole new family of genes is regulated by the same mechanism of alternative secondary RNA structures.

Tiling array study of MNNG treated Escherichia coli reveals a widespread transcriptional response.

The alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is known to trigger the adaptive response by inducing the ada-regulon - consisting of three DNA repair enzymes Ada, AlkB, AlkA and the enigmatic AidB. We have applied custom designed tiling arrays to study transcriptional changes in Escherichia coli following a MNNG challenge. Along with the expected upregulation of the adaptive response genes (ada, alkA and alkB), we identified a number of differentially expressed transcripts, both novel and annotated. This indicates a wider regulatory response than previously documented. There were 250 differentially-expressed and 2275 similarly-expressed unannotated transcripts. We found novel upregulation of several stress-induced transcripts, including the SOS inducible genes recN and tisAB, indicating a novel role for these genes in alkylation repair. Furthermore, the ada-regulon A and B boxes were found to be insufficient to explain the regulation of the adaptive response genes after MNNG exposure, suggesting that additional regulatory elements must be involved.

Antenatal HIV-1 RNA load and timing of mother to child transmission; a nested case-control study in a resource poor setting.

OBJECTIVE: To determine HIV-1 RNA load during the third trimester of pregnancy and evaluate its effect on in utero and intra-partum/postpartum transmissions in a breastfeeding population. DESIGN: A nested case-control study within a PMTCT cohort of antiretroviral therapy naive pregnant women and their infants. METHODS: A case was a mother who transmitted HIV-1 to her infant (transmitter) who was matched to one HIV-1 positive but non-transmitting mother (control). RESULTS: From a cohort of 691 pregnant women, 177 (25.6%) were HIV-1 positive at enrollment and from these 29 (23%) transmitted HIV-1 to their infants, 10 and 19 during in utero and intra-partum/postpartum respectively. Twenty-four mothers sero-converted after delivery and three transmitted HIV-1 to their infants. Each unit increase in log10 viral load was associated with a 178 cells/mm(3) and 0.2 g/dL decrease in TLC and hemoglobin levels, p = 0.048 and 0.021 respectively, and a 29% increase in the risk of transmission, p = 0.023. Intra-partum/postpartum transmitters had significantly higher mean viral load relative to their matched controls, p = 0.034. CONCLUSION: Antenatal serum HIV-1 RNA load, TLC and hemoglobin levels were significantly associated with vertical transmission but this association was independent of transmission time. This finding supports the rationale for preventive strategies designed to reduce vertical transmission by lowering maternal viral load.

Tiling array analysis of UV treated Escherichia coli predicts novel differentially expressed small peptides.

BACKGROUND: Despite comprehensive investigation, the Escherichia coli SOS response system is not yet fully understood. We have applied custom designed whole genome tiling arrays to measure UV invoked transcriptional changes in E. coli. This study provides a more complete insight into the transcriptome and the UV irradiation response of this microorganism. RESULTS: We detected a number of novel differentially expressed transcripts in addition to the expected SOS response genes (such as sulA, recN, uvrA, lexA, umuC and umuD) in the UV treated cells. Several of the differentially expressed transcripts might play important roles in regulation of the cellular response to UV damage. We have predicted 23 novel small peptides from our set of detected non-gene transcripts. Further, three of the predicted peptides were cloned into protein expression vectors to test the biological activity. All three constructs expressed the predicted peptides, in which two of them were highly toxic to the cell. Additionally, a remarkably high overlap with previously in-silico predicted non-coding RNAs (ncRNAs) was detected. Generally we detected a far higher transcriptional activity than the annotation suggests, and these findings correspond with previous transcription mappings from E. coli and other organisms. CONCLUSIONS: Here we demonstrate that the E. coli transcriptome consists of far more transcripts than the present annotation suggests, of which many transcripts seem important to the bacterial stress response. Sequence alignment of promoter regions suggest novel regulatory consensus sequences for some of the upregulated genes. Finally, several of the novel transcripts identified in this study encode putative small peptides, which are biologically active.

Evolution of human immunodeficiency virus type 1 serotypes in northern Tanzania: a retrospective study.

The HIV-1 epidemic in Tanzania is characterized by the circulation of heterogeneous virus subtypes. A retrospective study was conducted to determine the changing pattern of circulating HIV-1 subtypes in northern Tanzania. A peptide-binding enzyme immunoassay (PEIA) was employed to analyse 305 HIV-1 positive serum and plasma samples collected between 1985 and 2005. Samples were serotyped using synthetic peptides representing HIV-1 genotypes A-E derived from consensus gp120 V3 sequences. Plasma samples collected in 2005 were V3 genotyped for comparison with PEIA results. In 1985, serotypes A and D were co-circulating while serotype C was first detected in 1990. In 2001 and 2005, serotype C was the most prevalent, serotype A was stable, and serotype D was declining. PEIA is relatively rapid and simple to perform compared to molecular approaches, and is a valuable epidemiological tool in regions with limited resources. HIV-1 classification into serotypes based on antigenic V3 diversity may be a useful screening tool for the identification of HIV-1 variants with regard to diagnosis, treatment, disease progression and candidate vaccine development.

Prevalence of reverse transcriptase and protease mutations associated with antiretroviral drug resistance among drug-naïve HIV-1 infected pregnant women in Kagera and Kilimanjaro regions, Tanzania.

BACKGROUND: Access to antiretroviral drugs for HIV-1 infection has increased in sub-Saharan Africa (SSA) during the past few years. Mutations in the HIV-1 genome are often associated with treatment failure as indicated by viral replication and elevated levels of virus in the blood. Mutations conferring resistance to antiretroviral drugs are based on comparing gene sequences with corresponding consensus sequences of HIV-1 subtype B that represents only 10% of the AIDS pandemic. The HIV pandemic in SSA is characterized by high viral genetic diversity. Before antiretroviral drugs become more widely available, it is important to characterize baseline naturally occurring genetic mutations and polymorphisms associated with antiretroviral drug resistance among circulating HIV-1 subtypes. METHODS: The prevalence of mutations associated with antiretroviral drug resistance in protease (PR) and reverse transcriptase (RT) regions among antiretroviral treatment-naïve HIV-1 infected pregnant women was investigated in Bukoba (Kagera) and Moshi (Kilimanjaro) municipalities, Tanzania, between September and December 2005. The HIV-1 pol gene was amplified using primers recognizing conserved viral sequences and sequenced employing BigDye chemistry from 100 HIV-1 seropositive treatment-naïve pregnant women and 61 HIV-1 seropositive women who had received a single dose of Nevirapine (sdNVP). Positions 1-350 of the RT and 1-99 of the PR genes were analyzed for mutations based on the Stanford University HIV Drug Resistance Database. RESULTS: HIV-1 subtypes A, C, D, CRF10_CD and Unique Recombinant Forms (URF) were detected. Primary mutations associated with NRTI and NNRTI resistance were detected among 3% and 4% of treatment-naïve strains, respectively. Primary mutations associated with NRTI and NNRTI resistance were detected in 1.6% and 11.5% of women who had received sdNVP, respectively. None of the primary mutations associated with PI resistance was found. Polymorphisms detected in RT and PR sequences were mainly mutations that are found in the consensus sequences of non-B subtypes CONCLUSION: Based on the WHO HIV Drug Resistance Research Network Threshold of less than 5%, the baseline prevalence of primary mutations among treatment-naïve HIV-1 infected pregnant women in Kagera and Kilimanjaro regions was low. The significance of HIV-1 subtype B polymorphic positions with respect to antiretroviral resistance identified among the prevalent HIV-1 subtypes is unknown. More studies addressing the correlation between polymorphic mutations, antiretroviral resistance and clinical outcome are warranted in regions where non-B subtypes are prevalent.

Diversity of human immunodeficiency virus type 1 subtypes in Kagera and Kilimanjaro regions, Tanzania.

A strategy to prevent the spread of HIV-1 worldwide is complicated by the high genetic diversity of the virus. To gain a better understanding of the HIV-1 genetic diversity in Tanzania, a molecular epidemiological investigation was conducted in Kagera and Kilimanjaro regions. While several studies have addressed HIV-1 subtypes in Tanzania, this is the first study to describe the virus subtypes circulating in Kagera. The Kagera region is the epicenter of the HIV-1 epidemic in Africa, and it was therefore of interest to compare the prevalence of HIV subtypes in this region and Kilimanjaro. Blood samples were obtained from 246 HIV-1-infected pregnant women attending antenatal clinics. Plasma HIV-1 RNA was extracted, amplified, and sequenced in the env C2V3 and/or pol regions from 209 samples. Based on the analysis of env C2V3 and pol sequences, 47.4% had concordant subtypes, 19.1% were discordant indicating recombination, and for 33.5% sequences were obtained for only one region. The distribution HIV-1 subtypes based on the phylogenetic analysis of paired env C2V3/ pol sequences in Kagera region was A/A (27.8%), C/C (29.6%), D/D (16.7%), and unique recombinant forms (25.9%), and in Kilimanjaro region was A/A (32.9%), C/C (25.9%), D/D (10.6%), CRF10_CD (1.2%), and unique recombinant forms (29.4%). The env C2V3 subsubtype A2 and env C2V3/pol CRF10_CD were also observed indicating that these recombinants are circulating in Tanzania. The high diversity of HIV-1 subtypes and the high prevalence of recombinants demonstrated in this study necessitate expanded and continuous monitoring of the epidemic in Tanzania. The trend may have implications for current national control strategies against the HIV-1 epidemic.

Predicting non-coding RNA genes in Escherichia coli with boosted genetic programming.

Several methods exist for predicting non-coding RNA (ncRNA) genes in Escherichia coli (E.coli). In addition to about sixty known ncRNA genes excluding tRNAs and rRNAs, various methods have predicted more than thousand ncRNA genes, but only 95 of these candidates were confirmed by more than one study. Here, we introduce a new method that uses automatic discovery of sequence patterns to predict ncRNA genes. The method predicts 135 novel candidates. In addition, the method predicts 152 genes that overlap with predictions in the literature. We test sixteen predictions experimentally, and show that twelve of these are actual ncRNA transcripts. Six of the twelve verified candidates were novel predictions. The relatively high confirmation rate indicates that many of the untested novel predictions are also ncRNAs, and we therefore speculate that E.coli contains more ncRNA genes than previously estimated.