Alternative translation initiation augments the human mitochondrial proteome

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License.-- et al.Alternative translation initiation (ATI) is a mechanism of producing multiple proteins from a single transcript, which in some cases regulates trafficking of proteins to different cellular compartments, including mitochondria. Application of a genome-wide computational screen predicts a cryptic mitochondrial targeting signal for 126 proteins in mouse and man that is revealed when an AUG codon located downstream from the canonical initiator methionine codon is used as a translation start site, which we term downstream ATI (dATI). Experimental evidence in support of dATI is provided by immunoblotting of endogenous truncated proteins enriched in mitochondrial cell fractions or of co-localization with mitochondria using immunocytochemistry. More detailed cellular localization studies establish mitochondrial targeting of a member of the cytosolic poly(A) binding protein family, PABPC5, and of the RNA/DNA helicase PIF1α. The mitochondrial isoform of PABPC5 co-immunoprecipitates with the mitochondrial poly(A) polymerase, and is markedly reduced in abundance when mitochondrial DNA and RNA are depleted, suggesting it plays a role in RNA metabolism in the organelle. Like PABPC5 and PIF1α, most of the candidates identified by the screen are not currently annotated as mitochondrial proteins, and so dATI expands the human mitochondrial proteome.Cambridge University Commonwealth Trust, Fellowship (to L.K.); UK Medical Research Council. Funding for open access charge: UK Medical Research Council.Peer reviewe

The relationship between a child's postural stability and manual dexterity

The neural systems responsible for postural control are separate from the neural substrates that underpin control of the hand. Nonetheless, postural control and eye-hand coordination are linked functionally. For example, a stable platform is required for precise manual control tasks (e.g. handwriting) and thus such skills often cannot develop until the child is able to sit or stand upright. This raises the question of the strength of the empirical relationship between measures of postural stability and manual motor control. We recorded objective computerised measures of postural stability in stance and manual control in sitting in a sample of school children (n = 278) aged 3-11 years in order to explore the extent to which measures of manual skill could be predicted by measures of postural stability. A strong correlation was found across the whole sample between separate measures of postural stability and manual control taken on different days. Following correction for age, a significant but modest correlation was found. Regression analysis with age correction revealed that postural stability accounted for between 1 and 10 % of the variance in manual performance, dependent on the specific manual task. These data reflect an interdependent functional relationship between manual control and postural stability development. Nevertheless, the relatively small proportion of the explained variance is consistent with the anatomically distinct neural architecture that exists for 'gross' and 'fine' motor control. These data justify the approach of motor batteries that provide separate assessments of postural stability and manual dexterity and have implications for therapeutic intervention in developmental disorders.</p

Analysis of Replicating Mitochondrial DNA by In Organello Labeling and Two-Dimensional Agarose Gel Electrophoresis.

Our understanding of the mechanisms of DNA replication in a broad range of organisms and viruses has benefited from the application of two-dimensional agarose gel electrophoresis (2D-AGE). The method resolves DNA molecules on the basis of size and shape and is technically straightforward. 2D-AGE sparked controversy in the field of mitochondria when it revealed replicating molecules with lengthy tracts of RNA, a phenomenon never before reported in nature. More recently, radioisotope labeling of the DNA in the mitochondria has been coupled with 2D-AGE. In its first application, this procedure helped to delineate the "bootlace mechanism of mitochondrial DNA replication," in which processed mitochondrial transcripts are hybridized to the lagging strand template at the replication fork as the leading DNA strand is synthesized. This chapter provides details of the method, how it has been applied to date and concludes with some potential future applications of the technique.We thank Drs. Pohjoismäki and Jacobs for the EM image of the replicating mitochondrial DNA molecule, and the Medical Research Council for funding

Transcript availability dictates the balance between strand-asynchronous and strand-coupled mitochondrial DNA replication.

Mammalian mitochondria operate multiple mechanisms of DNA replication. In many cells and tissues a strand-asynchronous mechanism predominates over coupled leading and lagging-strand DNA synthesis. However, little is known of the factors that control or influence the different mechanisms of replication, and the idea that strand-asynchronous replication entails transient incorporation of transcripts (aka bootlaces) is controversial. A firm prediction of the bootlace model is that it depends on mitochondrial transcripts. Here, we show that elevated expression of Twinkle DNA helicase in human mitochondria induces bidirectional, coupled leading and lagging-strand DNA synthesis, at the expense of strand-asynchronous replication; and this switch is accompanied by decreases in the steady-state level of some mitochondrial transcripts. However, in the so-called minor arc of mitochondrial DNA where transcript levels remain high, the strand-asynchronous replication mechanism is instated. Hence, replication switches to a strand-coupled mechanism only where transcripts are scarce, thereby establishing a direct correlation between transcript availability and the mechanism of replication. Thus, these findings support a critical role of mitochondrial transcripts in the strand-asynchronous mechanism of mitochondrial DNA replication; and, as a corollary, mitochondrial RNA availability and RNA/DNA hybrid formation offer means of regulating the mechanisms of DNA replication in the organelle

Intercalibration of four spectrofluorometric protocols for measuring RNA/DNA ratios in larval and juvenile fish

The ratio of tissue RNA to DNA (R/D) is a widely used index of recent growth and nutritional condition in larval and juvenile fish. To date, however, no standard technique for measuring nucleic acids has been adopted. Because methodological details can affect the estimate of R/D, researchers using different analytical protocols have been unable to compare ratios directly. Here, we report on the results of an international interlaboratory calibration of 4 spectrofluorometric protocols to quantify nucleic acids. Replicate sets of 5 tissue samples and 2 standards (common standards) were supplied to each of 5 researchers for analysis with their own methods and standards. Two approaches were evaluated for mitigating the observed differences in values: 1) the use of common nucleic acid standards and 2) standardizing to a common slope ratio (slope of DNA standard curve/slope of RNA standard curve or mDNA/mRNA). Adopting common standards slightly reduced the variability among protocols but did not overcome the problem. When tissue R/Ds were standardized based on a common mDNA/mRNA slope ratio, the variance attributed to analytical protocol decreased dramatically from 57.1% to 3.4%. We recommend that the ratio of the slopes of the standard curves be provided to facilitate intercomparability of R/D results among laboratories using different spectrofluorometric methods for the analysis of nucleic acids in fish

Bash2py: A bash to Python translator

Abstract—Shell scripting is the primary way for programmers to interact at a high level with operating systems. For decades bash shell scripts have thus been used to accomplish various tasks. But Bash has a counter-intuitive syntax that is not well understood by modern programmers and is no longer adequately supported, making it now difficult to maintain. Bash also suffers from poor performance, memory leakage problems, and limited functionality which make continued dependence on it problematic. At the request of our industrial partner, we therefore developed a source-to-source translator, bash2py, which converts bash scripts into Python. Bash2py leverages the open source bash code, and the internal parser employed by Bash to parse any bash script. However, bash2py re-implements the variable expansion that occurs in Bash to better generate correct Python code. Bash2py correctly converts most Bash into Python, but does require human intervention to handle constructs that cannot easily be automatically translated. In our experiments on real-world open source bash scripts bash2py successfully translates 90 % of the code. Feedback from our industrial partner confirms the usefulness of bash2py in practice

Latewood Ring Width Reveals CE 1734 Felling Dates for Walker House Timbers In Tupelo, Mississippi, USA

Dendroarchaeology is under-represented in the Gulf Coastal Plain region of the United States (US), and at present, only three published studies have precision dated a collection of 18th–19th-century structures. In this study, we examined the tree-ring data from pine, poplar, and oak timbers used in the Walker House in Tupelo, Mississippi. The Walker House was constructed ca. the mid-1800s with timbers that appeared to be recycled from previous structures. In total, we examined 30 samples (16 pines, 8 oaks, and 6 poplars) from the attic and crawlspace. We cross-dated latewood ring growth from the attic pine samples to the period 1541–1734 (r = 0.52, t = 8.43, p \u3c 0.0001) using a 514-year longleaf pine (Pinus palustris Mill.) latewood reference chronology from southern Mississippi. The crawlspace oak samples produced a 57-year chronology that we dated against a white oak (Quercus alba L.) reference chronology from northeast Alabama to the period 1765–1822 (r = 0.36, t = 2.83, p \u3c 0.01). We were unable to cross-date the six poplar samples due to a lack of poplar reference chronologies in the region. Our findings have two important implications: (1) the pine material dated to 1734 represents the oldest dendroarchaeology-confirmed dating match for construction materials in the southeastern US, and (2) cross-dating latewood growth for southeastern US pine species produced statistically significant results, whereas total ring width failed to produce significant dating results

Storm-generated Holocene and historical floods in the Manawatu River, New Zealand

This paper reports the first reconstruction of storm-generated late Holocene and historical river floods in the North Island of New Zealand. The sedimentary infills of nine palaeochannels were studied in the lower alluvial reaches of the Manawatu River. Floods in these palaeochannels were recorded as a series of sand-rich units set within finer-grained fills. Flood chronologies were constrained using a combination of radiocarbon dating, documentary sources, geochemical markers, and palynological information. Flood units were sedimentologically and geochemically characterised using high resolution ITRAXTM 16 X-Ray Fluorescence (XRF) core scanning and laser diffraction grain-size analysis. The longest palaeoflood record extends back ca.3000 years. The temporal resolution and length of the Manawatu record reflects accommodation space for fluvial deposits, channel dynamics and mobility, and high sediment supply. Floods that occurred in the Manawatu during the mid-1800s at the time of European land clearance and in the first decade of the twentieth century appear to be among the largest recorded in the last 3000 year