Dominant-Negative Mutants of Ytm1p, an Essential Non-Ribosomal Protein in Saccharomyces cerevisae

Ribosome assembly is a complicated process involving the maturation of rRNA and it’s association with ribosomal proteins. Non-ribosomal proteins are also involved in coordinating the assembly of ribosomes by associating with the pre-rRNPs (ribonuclear protein particles) and then later dissociating. Ytm1p, an essential and conserved gene, was found to be involved in the maturation of the 66S pre-rRNP in both classic genetic studies and state of the art proteomic studies. Ytm1p is one of 17 known proteins involved in ribosome assembly that contains WD40 repeat structures. However, none of these proteins have been studied in great detail. Thus, studying the structure and function of Ytm1p can provide a model for the behavior of the WD40 repeat protein in multi-molecular complexes. Ytm1p, with its seven WD40 repeats, may be used as a scaffolding protein to nucleate and coordinate the assembly of protein-protein complexes or protein-RNA complexes used in the synthesis and maturation of the 66S pre-ribosomes. Specific sites on the protein may be involved in the scaffolding activity of Ytm1p. By mutating specific amino acids in the Ytm1p protein and studying the specific effects of the mutations, one can infer information on the function of the protein. Single point mutations were created in YTM1 using error-prone PCR and the genes were introduced into plasmids via gap repair. Mutant alleles of ytm1 were placed under the control of the inducible GAL1 promoter in order to screen for dominant-negative mutations. Seven mutants were identified in this screen: four strong dominant-negative mutants which inhibited growth of the yeast cells on media containing galactose, and three weaker mutants which only slowed growth on galactose media. These mutants were screened for defects in the synthesis of rRNA and will be screened for defects in polysome profiles and cell cycle progression. Characterizing the specific phenotypes of these dominant-negative mutations and their effects on the synthesis of the 60S ribosomal subunit will help to identify relationships between the structure of the Ytm1p and its function.

4th of july festivities

here are some fun things i did this past weekend:

cake concert

whirly ball


lazer tag

dinner at the winking lizard


fireworks from the roof of morewood


party at beth's house



spiderman 2

joe schmo 2 show!

overall a very good and exciting weekend. there was also lots of other good and exciting stuff left out of the above list but that's all good. if you want to see more pictures go to my andrew space.

and tonight will be cheeeeeeeeese! sampling at whole foods. that is also very exciting.

i just read a paper on ribosomal proteins that used dominant mutants. that is what i am doing in lab this summer. it's exciting to see someone else do similar studies as me and find big discoveries and publish a paper. i hope i can find exciting dominant negative mutants that lead to big discoveries about the function of my protein and lots of papers published in highly important journals. that would be cool. until then, i will keep plugging away at my transformations, pcr, site directed mutagenesis, and mutant screens. i'm hoping that some of my transformants will have mutants that cause the cells to die. good stuff.

fluffies say hello. and go eat cheeeeeeeeeeeese!

yay for transformants!!

my gap repair transformation actually worked!! i'm soooooo very very happy. that means that some of the mutated gene insert may have been incorporated into my gapped dna plasmid, and then the plasmid was taken up by the cells. the plasmid has a gene for an amino acid that the cells can't live without unless that amino acid is in the media they are growing in. so i plate them on media without that amino acid and they die unless they pick up the plasmid. there were lots of colonies growing on my plates, so lots of possible mutants to sort through and test. i spent a good amount of time today streaking out 60 of the transformants. crazy!

tonight is a cake concert at point park, which is also very good and exciting. if i leave work early i will be able to make some good food like cookies or chocolate covered strawberries to bring with me. yum yum!

yesterday i went on a photographic expedition through schenley park. i took one roll of film and then dropped the second one into a stream and destroyed it. so sad. now i have to figure out if i can get access to a darkroom this summer so i can develop the pictures. if not i will just have to wait until the fall. and my collection of undeveloped film will grow larger and larger. hooray!!

i am rather excited about whirley ball and the fourth of july fireworks and party this weekend. next week is going to be the best week of work ever. i have monday off for the holiday and thursday is the surp trip to kennywood. hopefully i can get cheap tickets somewhere. any suggestions anyone??

i started reading mirror mirror by gregory maguire (author of wicked and confessions of an ugly stepsister) at the beginning of the week, and now i am more than halfway finished with the book. it's really good. i like fairy tale stories and fantasy. fun stuff.

i also like fluffies.

and chocolate things.

but chocolate fluffies would be weird so don't even go there.

i have to go pour plates soon. and check on my site directed mutagenesis transformants. if they grew then this will be the best day of work ever!!