Polyamine sensing during antizyme mRNA programmed frameshifting

A key regulator of cellular polyamine levels from yeasts to mammals is the protein antizyme. The antizyme gene consists of two overlapping reading frames with ORF2 in the +1 frame relative to ORF1. A programmed +1 ribosomal frameshift occurs at the last codon of ORF1 and results in the production of full-length antizyme protein. The efficiency of frameshifting is proportional to the concentration of polyamines, thus creating an autoregulatory circuit for controlling polyamine levels. The mRNA recoding signals for frameshifting include an element 5' and a pseudoknot 3' of the shift site. The present work illustrates that the ORF1 stop codon and the 5' element are critical for polyamine sensing, whereas the 3' pseudoknot acts to stimulate frameshifting in a polyamine independent manner. We also demonstrate that polyamines are required to stimulate stop codon readthrough at the MuLV redefinition site required for normal expression of the GagPol precursor protein.     

A problem in multivariate analysis of codon usage data and a possible solution

Multivariate analyses are often used to identify major trends of variation in synonymous codon usage among genes. These analyses need to be performed on properly normalized codon usage data to avoid biases masking this synonymous variation, i.e., gene length, amino acid usage, and codon degeneracy; however, previous studies have failed to do so. In this paper, we demonstrate that the use of alternative normalized data (called 'relative adaptiveness' in the literature) can avoid all these biases and furthermore, can identify more trends of variation among genes, including GC-ending codon usage, GT-ending codon usage, and gene expression level.     

Position-specific incorporation of dansylated non-natural amino acids into streptavidin by using a four-base codon

Novel non-natural amino acids carrying a dansyl fluorescent group were designed, synthesized, and incorporated into various positions of streptavidin by using a CGGG four-base codon in an Escherichia coli in vitro translation system. 2,6-Dansyl-aminophenylalanine (2,6-dnsAF) was found to be incorporated into the protein more efficiently than 1,5-dansyl-lysine, 2,6-dansyl-lysine, and 1,5-dansyl-aminophenylalanine. Fluorescence measurements indicate that the position-specific incorporation of the 2,6-dnsAF is a useful technique to probe protein structures. These results also indicate that well-designed non-natural amino acids carrying relatively large side chains can be accepted as substrates of the translation system.     

Protein structure preference, tRNA copy number, and mRNA stem/loop content

From statistical analyses of protein sequences for humans and Escherichia coli we found that the messenger RNA segment of m-codons (for m=2 to 6) with average high tRNA copy number (TCN) (larger than approximately 10.5 for humans or approximately 1.95 for E. coli) preferably code for the alpha helix and that with low TCN (smaller than approximately 7.5 for humans or approximately 1.7 for E. coli) preferably code for coil. Between them there is an intermediate region without correlation to structure preference. For the beta strand the preference/ avoidance tendency is not obvious. All strong preference-modes of TCN for protein secondary structures have been deduced. The mutual interaction between two factors--protein secondary structural type and codon TCN--is tested by F distribution. A phenomenological model on the relation between structure preference and translational efficiency or accuracy is proposed. It is pointed out that the structure preference of codons is related to the distribution of mRNA stem/loop content in three TCN regions.     

On the role of the starved codon and the takeoff site in ribosome bypassing in Escherichia coli

Translating ribosomes can skip over stretches of messenger RNA and resume protein chain elongation after a "bypassed" region. We have previously shown that limitation for isoleucyl-tRNA can initiate a ribosome bypass when an AUA codon is in the ribosomal A-site. We have now generalized this effect to other "hungry" codons calling for four different limiting aminoacyl-tRNA species, suggesting that a pause at any A-site will have this effect. We have assessed bypassing in a large family of reporters with nearly every different triplet in the "takeoff site", i.e. the P-site on the 5' side of the hungry codon, and an identical "landing site" codon 16 nucleotides downstream. The different takeoff sites vary over a factor of 50 in bypassing proficiency. At least part of this variation appears to reflect stability of the codon Colon, two colons anticodon interaction at the takeoff site, as indicated by the following: (a) the bypassing proficiency of different tRNAs shows a rough correlation with the frequency of A Colon, two colons U as opposed to G Colon, two colons C pairs in the codon Colon, two colons anticodon association; (b) specific tRNAs bypass more frequently from codons ending in U than from their synonym ending in C; (c) an arginine tRNA with Inosine in the wobble position which reads CGU, CGC, and CGA bypasses much more frequently from the last codon than the first two synonyms.     

Intragenic codon bias in a set of mouse and human genes

To better conceptualize the mechanism underlying the evolution of synonymous codons, we have analysed intragenic codon usage in chosen "regions" of some mouse and human genes. We divided a given gene into two regions: one consisting of a trinucleotide repeat (TNR) and the other consisting of the "rest of the coding region" (RCR). Usually, a TNR is composed of a repetitive single codon, which may reflect its frequency in a gene. In contrast, a non-random frequency of a codon in the RCR versus TNR (or vice versa) of a gene should indicate a bias for that codon within the TNR. We examined this scenario by comparing codon frequency between the RCR and the cognate TNR(s) for a set of human and mouse genes. A TNR length of six amino acids or more was used to identify genes from the Genbank database. Twenty nine human and twenty one mouse genes containing TNRs coding for nine different amino acid runs were identified. The ratio of codon frequency in a TNR versus the corresponding RCR was expressed as "fold change" which was also regarded as a measure of codon bias (defined as preferential use either in TNR or in RCR). Chi-square values were then determined from the distribution of codon frequency in a TNR vs. the cognate RCR. At p<0.001, 22% and 27%, respectively, of human and mouse TNRs showed codon bias. Greater than 40% of the TNRs (29 out of 69 in human, and 18 of 42 in mouse) showed codon bias at p<0.05. In addition, we identify eight single-codon TNRs in mouse and ten in human genes. Thus, our results show intragenic codon bias in both mouse and human genes expressed in diverse tissue types. Since our results are independent of the Codon Adaptation Index (CAI) and starvation CAI, and since the tRNA repertoire in a cell or in a tissue is constant, our data suggest that other constraints besides tRNA abundance played a role in creating intragenic codon bias in these genes.     

黑曲霉中内切菊粉酶基因及其全合成基因在解脂耶氏酵母中表达的比较

利用酵母密码子偏爱性将黑曲霉(Aspergillus niger)中的内切菊粉酶(Endoinu linase)基因通过基因全合成的方式合成为酵母密码子偏爱性的内切菊粉酶基因。然后将原始和全合成的内切菊粉酶基因克隆到解脂耶氏酵母表达载体PINA1296上,得重组解脂耶氏酵母表达载体pHBM2020、pHBM2021,将两种质粒分别转化解脂耶氏酵母(Yarrowia lipolytica)CLIB725,筛选得到重组解脂耶氏酵母CLIB725(pHBM2020)、CLIB725(pHBM2021),将两种重组酵母摇瓶培养,经SDS-PAGE、测酶活检测表明两种基因在解脂耶氏酵母中都有表达,全合成菊粉酶比原始菊粉酶酶活要高。     

海洋真菌Fusariumsp.LD8岩藻多糖酶的液态发酵条件研究

通过对海洋真菌Fusariumsp.LD8产岩藻多糖酶发酵培养基组成及工艺条件的研究,得到较优培养基配方为:麸皮5%,海带粉2%,(NH4)2SO40.8%。以人工海水代替蒸馏水,接种量为3%,在恒温振荡器中以180r/min的速度振荡培养,岩藻多糖酶活可达2.10IU/mL。     

Spatial and temporal extinction dynamics in a freshwater cetacean

Geographical range contraction is a fundamental ecological characteristic of species population decline, but relatively little investigation has been conducted into general trends in the dynamic properties of range collapse. The Yangtze River dolphin or baiji (Lipotes vexillifer), probably the first large mammal species to have become extinct in over 50 years, was believed to have experienced major range collapse during its decline through progressive large-scale range contraction and fragmentation. This range-collapse model is challenged by a new dataset of 406 baiji last-sighting records collected from across the baiji''s historical range during an interview survey of Yangtze fishing communities. Although baiji regional abundance may have varied across its range, analyses of the extensive new sighting series provide comprehensive evidence that baiji population decline was not associated with any major contraction in geographical range across the middle–lower Yangtze drainage, even in the decade immediately before probable global extinction of the species. Extinction risk in baiji was therefore seemingly not related to evidence of range collapse. Baiji apparently underwent large-scale periodic and seasonal movements across their range, and we propose that range contraction and fragmentation may not be general biogeographic characteristics for declining populations of mobile species in connected landscapes.