Optimal Packaging of FIV Genomic RNA Depends upon a Conserved Long-range Interaction and a Palindromic Sequence within gag

The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5′ 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5′ and 3′ sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem-loop (SL2) and a small palindromic stem-loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8-5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5-3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV ψ.     

An integrated morphological and molecular approach to a new species description in the Trypanosomatidae: the case of Leptomonas podlipaevi n. sp., a parasite of Boisea rubrolineata (Hemiptera: Rhopalidae)

Leptomonas podlipaevi n. sp., a new trypanosomatid species, is described herein based on light microscopic, ultrastructural, and molecular phylogenetic data. The organism is pleomorphic both in host and culture, with two predominant forms-a typical promastigote with a long flagellum and a shorter promastigote with a small or barely extending flagellum. Several spliced leader RNA repeat sequences obtained from the original cultures and the clonal lines representing two types of cells were all nearly identical. These sequences formed a tight cluster in the neighbor-joining tree well separated from other trypanosomatid species. Glyceraldehyde phosphate dehydrogenase gene sequences were determined for L. podlipaevi and 10 previously described trypanosomatid species. Molecular phylogenetic analysis has demonstrated that the new species is most closely related to Leptomonas seymouri and Leptomonas pyrrhocoris. The analysis has also highlighted the polyphyly of the genus Leptomonas.     

Cloning and Nucleotide Sequence of the Gene Encoding a Serine Proteinase Inhibitor Named Marinostatin from a Marine Bacterium,Alteromonas sp. Strain B-10-31

The gene (mstI) encoding a serine proteinase inhibitor named marinostatin from marine Alteromonas sp. strain B-10-31 was cloned and its nucleotide sequence was analyzed. A short open reading frame of 192 bp encoded 63 amino acids with a molecular weight of 6,985. Furthermore, the initial product of marinostatin (marinostatin L) was purified and its amino acid sequence was analyzed. These results indicate that marinostatin is produced as a unique precursor consisting of the mature peptide and the leader peptide for an ATP-binding cassette (ABC) transporter, and furthermore the initial product of marinostatin is dehydrated and processed by proteolysis to give homologous forms of marinostatin.     

水稻黄矮弹状病毒的leader区和trailer区的序列分析及其体内转录物的鉴定

克隆及测定了水稻黄矮病毒基因组RNA的 3′端leader区和 5′端trailer区的序列 .结果表明 ,RYSV的 3′leader长 2 0 3个核苷酸 ,5′trailer长 191个核苷酸 .两者末端的 9个核苷酸完全互补 ,可形成弹状病毒基因组RNA的典型的锅柄结构 .与其他弹状病毒的leader和trailer相比较 ,RYSV的leader和trailer较长 ,除leader的 3′末端和trailer的 5′末端序列具有一定的保守性外 ,其余的核苷酸序列无明显的同源性 .用 3′RACE方法在感染RYSV的水稻中检测出leader的转录物 ,并证明该leaderRNA具有polyA尾巴 .这是继苦苣菜黄脉病毒的leaderRNA后第 2例弹状病毒leaderRNA带polyA尾巴的报道 .用类似的方法没有检测到带polyA尾巴的trailer的正链转录物 .     

Structure and dynamics of the M13 coat signal sequence in membranes by multidimensional high-resolution and solid-state NMR spectroscopy

The polypeptide corresponding to the signal sequence of the M13 coat protein and the five N-terminal residues of the mature protein was prepared by solid-phase peptide synthesis with a ¹⁵N isotopic label at the alanine-12 position. Multidimensional solution NMR spectroscopy and molecular modeling calculations indicate that this polypeptide assumes helical conformations between residues 5 and 20, in the presence of sodium dodecylsulfate micelles. This is in good agreement with circular dichroism spectroscopic measurement, which shows an α-helix content of approximately 42%. The α-helix comprises an uninterrupted hydrophobic stretch of ≤12 amino acids, which is generally believed to be too short for a stable transmembrane alignment in a biological bilayer. The monoexponential proton-deuterium exchange kinetics of this hydrophobic helical region is characterized by half-lives of 15–75 minutes (pH 4.2, 323 K). When the polypeptide is reconstituted into phospholipid bilayers, the broad anisotropy of the proton-decoupled ¹⁵N solid-state NMR spectroscopy indicates that the hydrophobic helix is immobilized close to the lipid bilayer surface at the time scale of ¹⁵N solid-state NMR spectroscopy (10⁻⁴ seconds). By contrast, short correlation times, immediate hydrogen-deuterium exchange as well as nuclear Overhauser effect crosspeak analysis suggest that the N and C termini of this polypeptide exhibit a mobile random coil structure. The implications of these structural findings for possible mechanisms of membrane insertion and translocation as well as for membrane protein structure prediction algorithms are discussed. © 1997 Wiley-Liss Inc.     

Nuclear Genome Sequence Survey of the Dinoflagellate Heterocapsa triquetra

ABSTRACT. Dinoflagellates have among the largest nuclear genomes known, but we know little about their contents or organisation. Given the interest in dinoflagellate ecology, cell biology, and evolutionary biology, there are many reasons to thoroughly investigate the contents of dinoflagellate genomes, but because of their large size the only thorough samples to date have relied on expressed sequence tag surveys to analyse cDNAs. To complement this, there are some studies of the physical properties of dinoflagellate chromosomes, but no direct survey of the nature of the sequences contained within them. To start to build a picture of the contents of these genomes, we have sequenced over 230,000 bp from the nuclear genome of Heterocapsa triquetra, which has been estimated to be 18–23 billion base pairs in total. The survey includes one putative gene with two relict spliced leaders, one putative pseudogene, and a small number of low‐complexity repeats, transposons, and other putative selfish elements, all of which account for about 5% of the survey. Another 5% of the survey was long, complex repeats, some highly represented. By far the greatest fraction of the survey (89.5%) is made up of non‐repeated sequence with no similarity to any other known sequence.