Biomedical subjects

Michelle Hanna

Publications and source records attributed to Michelle Hanna.

5 recordsLinked to original sources

PubMed2006

Isolation of nucleic acids.

Saccharomyces cerevisiae is an excellent model organism for the study of eukaryotic genetics. Easy manipulation of yeast DNA is essential to its role in research, and studies of gene expression or regulation require analysis of RNA. This chapter presents quick and straightforward methods to isolate genomic DNA, plasmid DNA, or RNA from yeast. The isolation protocols presented here, which utilize a glass bead method to break through the cell wall, will yield plasmid DNA of sufficient quality to transform into Escherichia coli, genomic DNA that can be digested with restriction enzymes for Southern blotting, or RNA for use in applications such as Northern blots.

DNA, Fungal↗

PubMed2006

Mutagenesis.

To identify new genes in an organism, a genetic approach can be used to screen for mutations that display a particular phenotype. Genotoxic agents, such as ultraviolet (UV) light, ionizing radiation, or chemicals can be used to randomly induce DNA lesions in the genome. Most efficient mutagenesis occurs when a mutagen confers a high frequency of mutations with low lethality, in the range of 10 to 50% survival. These mutations can be in the form of frameshifts, deletions, or rearrangements. To initiate a mutagenesis, a fresh subculture of cells grown into log phase is collected, washed, and resuspended in potassium phosphate buffer. The mutagen is added to the culture for a predetermined time, deactivated, and washed from the cells. The cells are allowed to recover from the treatment by incubating in liquid or on solid medium. Mutants can be isolated by screening individual colonies or by using direct selection of cells from the mutagenized cell population.

4-Nitroquinoline-1-oxide↗

PubMed2004

The TRAF6 RING finger domain mediates physical interaction with Ubc13.

Tumor necrosis factor receptor associated factor 6 (TRAF6) is an important signaling molecule involved in a diverse array of physiological processes. It has been proposed that TRAF6, a RING finger-containing protein, acts as a ubiquitin ligase (E3) and a target for Lys-63 linked polyubiquitination mediated by Ubc13-Uev, a ubiquitin conjugating (E2) complex. However, the physical interaction between TRAF6 and this E2 complex has not been reported. We used the yeast two-hybrid assay to demonstrate that TRAF6 indeed interacts with the E2 complex through its direct binding to Ubc13. Either a single Cys-to-Ser substitution within the TRAF6 RING finger domain or an amino acid substitution on the Ubc13 surface, that is predicted to interact with RING finger proteins, is able to abolish the interaction. In addition, we found that TRAF6 can interact with itself and this self-interaction domain is mapped to the N-terminus containing the RING finger motif. Based on this study and our previous Ubc13-Uev structural analysis, the interface of Ubc13-TRAF6 RING finger can be predicted.

Amino Acid Motifs↗

PubMed2004

Involvement of two endonuclease III homologs in the base excision repair pathway for the processing of DNA alkylation damage in Saccharomyces cerevisiae.

DNA base excision repair (BER) is initiated by DNA glycosylases that recognize and remove damaged bases. The phosphate backbone adjacent to the resulting apurinic/apyrimidinic (AP) site is then cleaved by an AP endonuclease or glycosylase-associated AP lyase to invoke subsequent BER steps. We have used a genetic approach in Saccharomyces cerevisiae to determine whether or not AP sites are blocks to DNA replication and the biological consequences if AP sites persist in the genome. We previously reported that yeast cells deficient in the two AP endonucleases (apn1 apn2 double mutant) are extremely sensitive to killing by a model DNA alkylating agent methyl methanesulfonate (MMS) and that this sensitivity can be reduced by deleting the MAG1 3-methyladenine DNA glycosylase gene. Here we report that in the absence of the AP endonucleases, deletion of two Escherichia coli endonuclease III homologs, NTG1 and NTG2, partially suppresses MMS-induced killing, which indicates that the AP lyase products are deleterious unless they are further processed by an AP endonuclease. The severe MMS sensitivity seen in AP endonuclease deficient strains can also be rescued by treatment of cells with the AP lyase inhibitor methoxyamine, which suggests that the product of AP lyase action on an AP site is indeed an extremely toxic lesion. In addition to the AP endonuclease interactions, deletion of NTG1 and NTG2 enhances the mag1 mutant sensitivity to MMS, whereas overexpression of MAG1 in either the ntg1 or ntg2 mutant severely affects cell growth. These results help to delineate alkylation base lesion flow within the BER pathway.

Alkylation↗

PubMed2002

A homologue of CROC-1 in a ciliated protist (Sterkiella histriomuscorum) testifies to the ancient origin of the ubiquitin-conjugating enzyme variant family.

Resting cysts of Sterkiella histriomuscorum (Ciliophora, Oxytrichidae) have been shown to contain messenger RNA, one of which codes for a protein significantly similar to CROC-1. CROC-1 is a human regulatory protein capable of transactivating the promoter of c-fos and belongs to a newly characterized family of ubiquitin-conjugating enzyme (E2) variants (UEV). We have determined the corresponding macronuclear gene sequence, which is the first protistan UEV sequence available. The phylogenetic analysis indicates the deep separation and solid clustering of all the UEV sequences within the E2 tree showing the ancient origin of these regulatory genes and their high structural conservation during evolution. Furthermore, overexpression of the ciliate UEV is able to rescue the Saccharomyces cerevisiae mms2 null mutant from killing by DNA damaging agents, implying that the UEV family proteins are functionally conserved. In S. histriomuscorum, expression of UEV is correlated with the growth of the cells as transcripts are present in excysting and vegetative cells but are rapidly down-regulated during starvation. These data support the high conservation of the UEV family in eukaryotes, and a regulatory role of the gene is discussed in relation to known functions of UEVs. This analysis may promote the search for homologues of other regulatory genes (metazoan regulators of differentiation) in ciliates.

Amino Acid Sequence↗