Using the fluorescence decay of 2-aminopurine to investigate conformational change in the recognition sequence of the EcoRV DNA-(adenine-N6)-methyltransferase on enzyme binding

The EcoRV DNA methyltransferase methylates the first adenine in the GATATC recognition sequence. It is presumed that methylation proceeds by a nucleotide flipping mechanism but no crystal structure is available to confirm this. A popular solution-phase assay for nucleotide flipping employs the fluorescent adenine analogue, 2-aminopurine (2AP), substituted at the methylation target site; a substantial increase in fluorescence intensity on enzyme binding indicates flipping. However, this appeared to fail for M.EcoRV, since 2AP substituted for the non-target adenine in the recognition sequence showed a much greater intensity increase than 2AP at the target site. This anomaly is resolved by recording the fluorescence decay of 2AP which shows that the target 2AP is indeed flipped by the enzyme, but its fluorescence is quenched by interaction with aromatic residues in the catalytic site, whereas bending of the duplex at the non-target site alleviates inter-base quenching and exposes the 2AP to solvent.     

Evaluation of dried distillers' grains with solubles (DDGS) as a protein source for broilers

Male broiler chicks (n=120) were fed diets containing 0, 5, 10 or 15% dried distillers' grains with solubles (DDGS) from the 12th day up to the end of fattening (day 35). During this period feed intake, weight gain and excreta quality (pH, DM) were tested. A digestibility trial was carried out on four birds from each group on the last five days of the experiment to determine the digestibility of organic matter and CP of the different diets. The protein digestibility was evaluated using three different methods; uric acid correction, alpha-amino-N and amino acid-N. There were no significant effects of increased DDGS levels on feed intake, weight gain, excreta quality or digestibility of CP and organic matter. However, feed conversion showed a tendency to decline at the highest DDGS level (15%). Digestibility of DDGS protein was estimated to be 77%. There was no significant difference between uric acid and alpha-amino-N method, but both methods had a significantly lower CP digestibility than amino acid-N. The present results indicate that DDGS can be used as a protein source in diets for fattening broilers up to 10-15%.     

MMDB: Entrez’s 3D-structure database

Three-dimensional structures are now known within many protein families and it is quite likely, in searching a sequence database, that one will encounter a homolog with known structure. The goal of Entrez’s 3D-structure database is to make this information, and the functional annotation it can provide, easily accessible to molecular biologists. To this end Entrez’s search engine provides three powerful features. (i) Sequence and structure neighbors; one may select all sequences similar to one of interest, for example, and link to any known 3D structures. (ii) Links between databases; one may search by term matching in MEDLINE, for example, and link to 3D structures reported in these articles. (iii) Sequence and structure visualization; identifying a homolog with known structure, one may view molecular-graphic and alignment displays, to infer approximate 3D structure. In this article we focus on two features of Entrez’s Molecular Modeling Database (MMDB) not described previously: links from individual biopolymer chains within 3D structures to a systematic taxonomy of organisms represented in molecular databases, and links from individual chains (and compact 3D domains within them) to structure neighbors, other chains (and 3D domains) with similar 3D structure. MMDB may be accessed at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Structure.     

Transposon Tn21, Flagship of the Floating Genome

The transposon Tn21 and a group of closely related transposons (the Tn21 family) are involved in the global dissemination of antibiotic resistance determinants in gram-negative facultative bacteria. The molecular basis for their involvement is carriage by the Tn21 family of a mobile DNA element (the integron) encoding a site-specific system for the acquisition of multiple antibiotic resistance genes. The paradigm example, Tn21, also carries genes for its own transposition and a mercury resistance (mer) operon. We have compiled the entire 19,671-bp sequence of Tn21 and assessed the possible origins and functions of the genes it contains. Our assessment adds molecular detail to previous models of the evolution of Tn21 and is consistent with the insertion of the integron In2 into an ancestral Tn501-like mer transposon. Codon usage analysis indicates distinct host origins for the ancestral mer operon, the integron, and the gene cassette and two insertion sequences which lie within the integron. The sole gene of unknown function in the integron, orf5, resembles a puromycin-modifying enzyme from an antibiotic producing bacterium. A possible seventh gene in the mer operon (merE), perhaps with a role in Hg(II) transport, lies in the junction between the integron and the mer operon. Analysis of the region interrupted by insertion of the integron suggests that the putative transposition regulator, tnpM, is the C-terminal vestige of a tyrosine kinase sensor present in the ancestral mer transposon. The extensive dissemination of the Tn21 family may have resulted from the fortuitous association of a genetic element for accumulating multiple antibiotic resistances (the integron) with one conferring resistance to a toxic metal at a time when clinical, agricultural, and industrial practices were rapidly increasing the exposure to both types of selective agents. The compendium offered here will provide a reference point for ongoing observations of related elements in multiply resistant strains emerging worldwide.     

东亚飞蝗hunchback基因在卵子形成和胚胎发育过程中的原位表达

hb（hunchback）基因是昆虫胚胎前后轴模式形成的关键基因．对东亚飞蝗（Locusta migratoria manilensis,Meyen）hb基因的功能已有报道,但其表达模式还不清楚．为了研究胁基因在东亚飞蝗卵子形成和胚胎发育过程中的时空表达情况,本研究采用免疫组化方法在蛋白质水平上检测了hb基因的时空表达模式．在卵子形成过程中,hb基因局限在卵细胞核区中表达,随着卵子的发育逐渐移至卵细胞的后端;卵受精后,核区里的Hb蛋白向外扩散,在卵后端形成浓度梯度;胚盘期,hb基因在胚盘中央呈带状表达;胚盘分化为原头和原躯干后,表达条带变宽,并呈现出梯度表达,该表达区域将形成颌、胸部的部分体节;随着腹节开始形成,hb基因在颌胸部的表达逐渐减弱,而在腹部后端的“生长区”表达,并呈现出不连续性．经比较,hb易基因在昆虫颌胸部的表达较为保守,而在卵子形成过程中和腹部的表达具有较大的变异性．与黑腹果蝇等长胚带昆虫相比,东亚飞蝗hb基因在体节形成的基因级联调控中具有更重要、更直接的调控作用．