Inhibition of in vitro and ex vivo translation by a transplatin-modified oligo(2'-O-methylribonucleotide) directed against the HIV-1 gag-pol frameshift signal.

A 2'-O-methylribooligonucleotide containing a G1.U.G3 triad modified by trans-diamminedichloro-platinum(II) was targeted to the RNA region responsible for the gag-pol frameshifting during translation of the HIV-1 mRNA. The binding of the platinated oligonucleotide to its target RNA induced a rearrangement of the (G1, G3)-intrastrand crosslink, leading to the formation of an intermolecular oligonucleotide-RNA G-A crosslink. This resulted in the selective arrest of translation of a luciferase gene placed downstream of the HIV-1 frameshift signal both in a cell-free extract (rabbit reticulocyte lysate) and in RNA-transfected cells. A specific inhibition of luciferase activity was still observed when the oligonucleotide-RNA complex was not pre-formed prior to either translation or transfection. Moreover, a selective inhibition was also observed when the oligonucleotide and the plasmid DNA encoding the luciferase and bearing the RNA gag- pol frameshifting signal were co-transfected in NIH 3T3 cultured cells. Therefore the intra-strand-->interstrand conversion of the platinum crosslink kinetically competes with the translation machinery and blocks the polypeptide elongation. These transplatin-modified oligonucleotides which operate within a live cell on a 'real-time' basis and do not need an external triggering signal constitute a promising new class of selective reactive probes.

Four repeat high-mol-wt MAP2 forms in rat dorsal root ganglia.

The high-mol-wt forms of brain microtubule-associated protein 2 (MAP2a/b) segregate within the dendrites during neuronal differentiation, whereas a low-mol-wt variant, MAP2c, is distributed within all the neuronal domains. Both MAP2b and MAP2c contain three tubulin binding repeats, whereas another low-mol-wt form, MAP2d, contains four repeats. Since high-mol-wt MAP2 species with four repeats have been cloned so far only from the sensory ND cell line, we have studied in this work the high-mol-wt forms expressed by dorsal root ganglia (DRG). Different clones obtained from PCR amplification products of portions of the C-terminal and of the 3' end of the middle MAP2b domains contained either three or four tubulin binding repeats at adult stages and only three postnatally. In adulthood, two exons located at the 3' end of the MAP2b middle domain were missing in several clones: exon 10 within clones with three or four repeats, exon 11 only within those containing four repeats. Several other clones obtained from PCR amplification products of portions of the N-terminal and of the 5' end of the middle MAP2b domains revealed exons 7A and 8. In contrast, Northern analysis revealed exon 8 but not exon 7A, which is probably expressed in trace amounts in the DRG. In this article, we have identified for the first time high-mol-wt MAP2 transcripts containing four tubulin binding repeats that seem to be expressed only by the DRG and also differing from brain MAP2b by a number of other exons.

Hexose uptake in Trypanosoma cruzi: structure-activity relationship between substrate and transporter.

The gene encoding a hexose transporter, TcrHt1, from Trypanosoma cruzi has been functionally expressed in mammalian Chinese hamster ovary cells. Kinetic parameters of the heterologously expressed protein are very similar to those of the transporter identified in T. cruzi epimastigotes, confirming that TcrHT1 is the major transporter functioning in these parasites. A detailed analysis of substrate recognition using analogues of D-glucose substituted at each carbon position has been performed. The glucose transporter of T. cruzi does not recognize C-3 or C-6 analogues of D-glucose, whereas these analogues were recognized by the glucose transporter of bloodstream-form T. brucei. As for other kinetoplastid transporters, but in stark contrast to the mammalian GLUT family, TcrHT1 can also transport D-fructose, with relatively high affinity (Km = 0.682 +/- 0.003 mM). Amino acid side-chain-modifying reagents were also used to identify residues of the transporter present at the substrate-binding site. While specific modifiers of cysteine, histidine and arginine all inhibited catalytic activity, protection using substrate was only observed using the arginine-specific reagent, phenylglyoxal. Reagents which modify lysine residues had no effect on transport.

New forms of HMW MAP2 are preferentially expressed in the spinal cord.

The high molecular weight forms of microtubule-associated protein 2 (MAP2a and b) play a central role in the specification of dendrites. RT-PCR amplification of a portion of the N-terminal and middle MAP2b domains of rat spinal cord cDNAs allowed identification of new variants containing both exon 8 (246 bp) and a new exon, 7A (237 bp), located at the beginning of the middle MAP2b region. The brain and the spinal cord express transcripts containing exon 8, whereas exon 7A alone or exons 7A+8 were detected, whatever the developmental stage, only in the spinal cord.

Characterization of glucose transport and cloning of a hexose transporter gene in Trypanosoma cruzi.

A gene from Trypanosoma cruzi, TcrHT1, which encodes a member of the glucose transporter superfamily has been cloned. The gene is similar in sequence to the T. brucei hexose transporter THT1 and the Leishmania transporter Pro-1 and is present in the T. cruzi genome as a cluster of at least eight tandemly reiterated copies. Northern blot analysis revealed two mRNA transcripts which differ in size with respect to their 3' untranslated regions. When injected with in vitro transcribed TcrHT1 mRNA, Xenopus oocytes express a hexose transporter with properties similar to those of T. cruzi. Glucose transport in T. cruzi is mediated via a carrier with unique properties when compared with the other glucose transporters already characterized among the Kinetoplastida. It is a facilitated transporter with a high affinity for D-glucose (Km = 84.1 +/- 7.9 microM and Vmax = 46 +/- 9.4 nmol/min per mg of protein) that shares with other kinetoplastid hexose transporters the ability to recognize D-fructose, which distinguishes these carriers from the human erythrocyte glucose transporter GLUT1.