Biomedical subjects

Michael N Guerini

Publications and source records attributed to Michael N Guerini.

3 recordsLinked to original sources

PubMed2002

Genetic complementation in apicomplexan parasites.

A robust forward genetic model for Apicomplexa could greatly enhance functional analysis of genes in these important protozoan pathogens. We have developed and successfully tested a genetic complementation strategy based on genomic insertion in Toxoplasma gondii. Adapting recombination cloning to genomic DNA, we show that complementing sequences can be shuttled between parasite genome and bacterial plasmid, providing an efficient tool for the recovery and functional assessment of candidate genes. We show complementation, gene cloning, and biological verification with a mutant parasite lacking hypoxanthine-xanthine-guanine phosphoribosyltransferase and a T. gondii cDNA library. We also explored the utility of this approach to clone genes based on function from other apicomplexan parasites using Toxoplasma as a surrogate. A heterologous library containing Cryptosporidium parvum genomic DNA was generated, and we identified a C. parvum gene coding for inosine 5-monophosphate-dehydrogenase (IMPDH). Interestingly, phylogenetic analysis demonstrates a clear eubacterial origin of this gene and strongly suggests its lateral transfer from epsilon-proteobacteria. The prokaryotic origin of this enzyme might make it a promising target for therapeutics directed against Cryptosporidium.

Animals↗

PubMed2002

Isolation of a Toxoplasma gondii cyclin by yeast two-hybrid interactive screen.

GAL4-based yeast two-hybrid cDNA libraries from Toxoplasma gondii RH strain were constructed and screened for interactors of a putative T. gondii cdc2-related kinase, TgCRK2. A screen of 3.2 million transformants yielded a single yeast clone that harbored a protein fusion capable of specifically interacting with TgCRK2. Sequencing revealed the cDNA insert (TgCYC1) had homology to the cyclin class of proteins. The TgCYC1 cDNA fragment was used to probe a conventional T. gondii cDNA library and a 2.65 kb cDNA coding for a predicted protein of 582 amino acids was obtained. Based on comparison with a 5'-RACE product from tachyzoite mRNA, the 2.65 kb cDNA for TgCYC1 appeared to be complete. TgCYC1 had the highest similarity to Plasmodium falciparum CYC1 and displayed sequence characteristics that place it in the cyclin H class of eukaryotic cyclins. In synchronous tachyzoite populations the level of TgCYC1 mRNA was unchanged indicating it is not cell cycle regulated at the mRNA level. TgCYC1 rescues the G(1)/S cyclin cell cycle defect in S. cerevisiae strain DL1 demonstrating that this apicomplexan cyclin can function in an established heterologous model system.

Amino Acid Sequence↗

PubMed1999

Resistance of Capsicum annuum 'Avelar' to pepper mottle potyvirus and alleviation of this resistance by co-infection with cucumber mosaic cucumovirus are associated with virus movement.

Capsicum annuum cv. Avelar plants resist systemic infection by the Florida isolate of pepper mottle potyvirus (PepMoV-FL). Immuno-tissue blot analysis for detection of PepMoV-FL infection in selected stem segments revealed that virus moved down the stem in external phloem, and, over time, accumulated to detectable levels throughout stem sections (appearing to accumulate in external and internal phloem) taken from below the inoculated leaf. At 21 days post-inoculation, PepMoV-FL was detected in stem segments one or two internodes above the inoculated leaf; however, no virus was observed in internal phloem in stem segments beyond these internodes. In contrast to these observations, PepMoV-FL was detected in the internal phloem of all internodes of the stem located above the inoculated leaf, with subsequent movement into non-inoculated leaves, in Avelar plants co-infected with PepMoV-FL and cucumber mosaic cucumovirus (CMV-KM). No apparent enhancement of PepMoV-FL accumulation occurred in protoplasts inoculated with PepMoV-FL alone versus a mixed inoculum of PepMoV-FL and CMV-KM. These findings confirm earlier observations that potyvirus movement up the stem of Capsicum species occurs via internal phloem. It is also shown that PepMoV-FL does not accumulate to detectable levels in internal phloem in the stem of Avelar plants, thereby limiting its movement to within the inoculated leaf and lower portions of the stem; however, co-infection of Avelar plants with CMV-KM alleviates this restricted movement, allowing PepMoV-FL to invade young tissues systemically.

Biological Assay↗