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Mol Biol Evol. 2000 Oct;17(10):1435-45.
Cirripede phylogeny using a novel approach: molecular morphometrics.

Billoud B, Guerrucci MA, Masselot M, Deutsch JS.

Atelier de BioInformatique, Service Commun de Bio-Systematique, Universite Pierre et Marie Curie, Paris, France. bernard.billounv.jussieu.fr

We present a new method using nucleic acid secondary structure to assess phylogenetic relationships among species. In this method, which we term "molecular morphometrics," the measurable structural parameters of the molecules (geometrical features, bond energies, base composition, etc.) are used as specific characters to construct a phylogenetic tree. This method relies both on traditional morphological comparison and on molecular sequence comparison. Applied to the phylogenetic analysis of Cirripedia, molecular morphometrics supports the most recent morphological analyses arguing for the monophyly of Cirripedia sensu stricto (Thoracica + Rhizocephala + Acrothoracica). As a proof, a classical multiple alignment was also performed, either using or not using the structural information to realign the sequence segments considered in the molecular morphometrics analysis. These methods yielded the same tree topology as the direct use of structural characters as a phylogenetic signal. By taking into account the secondary structure of nucleic acids, the new method allows investigators to use the regions in which multiple alignments are barely reliable because of a large number of insertions and deletions. It thus appears to be complementary to classical primary sequence analysis in phylogenetic studies.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11018151&dopt=Abstract

J Microbiol Methods. 2000 Oct;42(2):203-7.
The addition of low numbers of 3' thymine bases can be used to improve the hybridization signal of oligonucleotides for use within arrays on nylon supports.

Brown TJ, Anthony RM.

Department of Microbiology, St. Thomas Hospital, Guy's and St. Thomas' Hospital Trust, London SE1 7EH, UK. timothy.browcl.ac.uk

Oligonucleotide arrays can be used for the analysis of microbial nucleic acid. The addition of high numbers of dTTP to the 3' ends of oligonucleotides using terminal transferase has been shown to facilitate membrane binding. This paper demonstrates low numbers of thymine bases added to the 3' end of oligonucleotides during synthesis can improve hybridisation signal intensity where the signal seen with the unmodified oligonucleotides is poor. Thus, the addition of variable numbers of thymine bases to different oligonucleotides allows the production of oligonucleotide arrays producing strong interpretable hybridisation signals.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11018277&dopt=Abstract

Biochimie. 2000 Aug;82(8):693-704.
Overproduction and improved strategies to purify the threenative forms of nuclease-free HU protein.

Pellegrini O, Oberto J, Pinson V, Wery M, Rouviere-Yaniv J.

Laboratoire de Physiologie Bacterienne, CNRS, UPR 9073, Institut de Biologie Physico-Chimique, 13, rue Pierre-et-Marie-Curie, 75005, Paris, France.

The heterodimeric HU protein was isolated from Escherichia coli as one of the most abundant DNA binding proteins associated with the bacterial nucleoid. HUalphabeta is composed of two very homologous subunits, but HU can also be present in E. coli under its two homodimeric forms, HUalpha(2) and HUbeta(2). This protein is conserved either in its heterodimeric form or in one of its homodimeric forms in all bacteria, in plant chloroplasts and in some viruses. HU can participate, like the histones, in the maintenance of DNA supercoiling and in DNA condensation. This protein which does not recognize any specific sequence on double-stranded DNA, has been shown to bind specifically to cruciform DNA as does the eukaryotic HMG1 protein and to a series of structures which are found as intermediates of DNA repair, e.g., nick, gap, 3'overhang, etc. The strong binding of HU to these diverse DNA structures could explain, in part at least, its pleiotropic role in the bacterial cell. To understand all the facets of its interactions with nucleic acids, it was necessary to develop a procedure which allowed the purification of the three forms of HU under their native form and without the nuclease activity strongly associated with the protein. We describe here such a procedure as well as demonstrating that the three histidine-tagged HUs we have produced, have conserved the binding characteristics of native HUs. Interestingly, by two complementation tests, we show that the histidine-tagged HUs are fully active in vivo.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11018285&dopt=Abstract

Biochim Biophys Acta. 2000 Sep 29;1481(2):381-94.
Fluorescence and nucleic acid binding properties of the human T-cell leukemia virus-type 1 nucleocapsid protein.

Morcock DR, Kane BP, Casas-Finet JR.

AIDS Vaccine Program, SAIC Frederick, National Cancer Institute - Frederick Cancer Research and Development Center, Bldg. 535, 4th floor, P.O. Box B, Frederick, MD 21702, USA.

We used intrinsic tryptophan fluorescence to study the nucleocapsid protein from human T-cell leukemia virus-type one, HTLV-1 p15, an 85-amino-acid protein with two Trp-containing zinc-finger motifs. Fluorescence spectra suggested an interaction between the two zinc fingers and another interaction involving the C-terminal tail and the zinc fingers. Titrations with nucleic acid revealed similar, sub-micromolar affinity for poly(dT) and poly(U) in 1 mM sodium phosphate, pH 7. Double-stranded DNA bound an order of magnitude weaker, suggesting helix-destabilizing activity. Base preference of p15 was T approximately U>I approximately C approximately G>A; affinity spanned about one order of magnitude. HTLV-1 p15 bound weaker and with less variation than reported values for either human or simian immunodeficiency virus homologues. The low affinity of p15 for nonspecific nucleic acids distinguishes it from other nucleocapsid proteins, and may suggest its involvement in additional steps of the virus life cycle other than RNA packaging.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11018730&dopt=Abstract

J Clin Lab Anal. 2000;14(5):238-45.
Evaluation of Henes-PCR assay for Mycobacterium detection in different clinical specimens from patients with or without tuberculosis-associated HIV infection.

Hernandez Abanto SM, Hirata MH, Hirata RD, Mamizuka EM, Schmal M, Hoshino-Shimizu S.

Department of Clinical and Toxicological Analyses, Faculty of Pharmaceutical Sciences, Sao Paulo University, Sao Paulo, Brazil.

The need for early diagnosis of tuberculosis, particularly in HIV-infected patients, requires the development of diagnosis methods that have a high sensitivity and specificity, as does the nucleic acid-based technology. With the purpose of improving the detection of mycobacterium in different clinical samples, we proposed and evaluated an assay based on nucleic acid-amplification: heminested-PCR (Henes-PCR). The procedure was designed to identify Mycobacterium spp., M. tuberculosis complex (MTC), and M. avium complex (MAC), although it has the potential to include more primers for the identification of other species. Analytical and clinical evaluation of Henes-PCR was performed by analysis of reference strains and 356 clinical specimens from 246 patients with pulmonary and meningitis tuberculosis and unrelated infections, including 142 HIV-infected individuals. Ninety-three percent (199) positive and 100% (143) negative results were obtained in specimens from patients with tuberculosis and non-tuberculosis infection, respectively. The overall sensitivity of Henes-PCR was 93.4%, specificity was 100%, positive and negative predictive values were 100 and 91.1%, respectively. Sensitivity and negative predictive value of Henes-PCR were significantly higher than culture procedure for microscopy-negative specimens. Even though frequency of HIV infection was higher in patients with tuberculosis, diagnostic parameters of Henes-PCR were similar between HIV-positive and HIV-negative patients. MTB was identified in 194 (98%) specimens while MAC was detected in 5 (2%) specimens. These findings suggest that Henes-PCR is a useful test for rapid detection of mycobacterium in clinically suspected cases of tuberculosis with smear-negative results. 2000 Wiley-Liss, Inc.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11018803&dopt=Abstract

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