A molecular evidence for the presence of methylobacterial-type Fe siderophore receptor in Celosia cristata

The presence of efficient iron-uptake bacteria was predicted to be localized as endosymbionts within the leaves of <I>Celosia cristata</I>, a well known iron-rich plant. On the other hand, the symbiotic methylobacterium having a distinctive pink pigmentation was suggested to be more likely in the leaves of pink-colored plants. These considerations were experimented by priming a cDNA fragment containing methylobacterial-type Fe siderophore receptor domain from <I>Celosia</I> leaf cDNA population. Since no detectable homologue was found in plant species sequenced to date, the presence of a Fe-efficient methylobacterium endosymbiosis was reliably predicted in <I>Celosia</I> plant. This is the first report that may lead to the way for future studies on molecular interactions between high iron content pink-colored plants and iron-efficient pink-pigmented bacteria. Corresponding cDNA sequence was submitted to EMBL databases under accession number FM955594.

Ribavirin Reveals a Lethal Threshold of Allowable Mutation Frequency for Hantaan Virus

ABSTRACT The broad spectrum of antiviral activity of ribavirin (RBV) lies in its ability to inhibit IMP dehydrogenase, which lowers cellular GTP. However, RBV can act as a potent mutagen for some RNA viruses. Previously we have shown a lack of correlation between antiviral activity and GTP repression for Hantaan virus (HTNV) and evidence for RBV's ability to promote error-prone replication. To further explore the mechanism of RBV, GTP levels, specific infectivity, and/or mutation frequency was measured in the presence of RBV, mycophenolic acid (MPA), selenazofurin, or tiazofurin. While all four drugs resulted in a decrease in the GTP levels and infectious virus, only RBV increased the mutation frequency of viral RNA (vRNA). MPA, however, could enhance RBV's mutagenic effect, which suggests distinct mechanisms of action for each. Therefore, a simple drop in GTP levels does not drive the observed error-prone replication. To further explore RBV's mechanism of action, we made a comprehensive analysis of the mutation frequency over several RBV concentrations. Of importance, we observed that the viral population reached a threshold after which mutation frequency did not correlate with a dose-dependent decrease in the level of vRNA, PFU, or [RTP]/[GTP] (where RTP is ribavirin-5′-triphosphate) over these same concentrations of RBV. Modeling of the relationship of mutation frequency and drug concentration showed an asymptotic relationship at this point. After this threshold, approximately 57% of the viral cDNA population was identical to the wild type. These studies revealed a lethal threshold, after which we did not observe a complete loss of the quasispecies structure of the wild-type genome, although we observed extinction of HTNV.

PCR-generated cDNA libraries from reduced numbers of mouse oocytes

SummaryWe describe a rapid and reproducible method for cloning cDNA amplified from 10 mouse oocytes. The procedure consists in priming cDNA synthesis from a crude cellular extract using an oligo d(T) containing primer and submitting the size-limited cDNA first strand to poly(dG) tailing. The whole cDNA population is then polymerase chain reaction (PCR) amplified using two primers complementary to oligo d(A) and oligo d(G) ends of the cDNA. In this procedure no purification steps are required. We obtained about 5 ×106 clones from 10 oocytes. Screening of the library showed that the relative abundance of the transcripts was preserved during amplification and cloning and that the procedure allows cloning of low-abundance sequences at least as rare as 0.008% of the mRNA. The repeatable generation of representative cDNA libraries from reduced numbers of oocytes or embryos should open new opportunities for obtaining genetic information from mammalian preimplantation embryos.