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Analysis of small proteins in Saccharomyces cerevisiae
Başlık:
Analysis of small proteins in Saccharomyces cerevisiae
Yazar:
Knight, David Robert, author.
ISBN:
9780438043756
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (252 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 76-08C.
Özet:
The availability of complete genome sequences is changing the way that biology is studied. Databases of the genetic complement are central to these new studies, where coding sequence is identified from genomes in a number of ways including sequence length, coding bias, Markov chain statistics and cross-species homology searches. All of these methods depend upon sufficient sequence to make statistical comparisons relevant or the previous identification of a representative number of similar genes. However statistical analyses have lower relevance for small genes and there are technical difficulties in analysing their products. The premise of this project is that small genes (<100 codons) and their products are under-represented in these databases. As not all potential coding sequences are transcribed and not all transcripts are translated the existence of small genes was investigated by the study of their protein products. There were two aims to this study, firstly to develop methods to profile small proteins in order to gauge their abundance and secondly to identify a representative number of these proteins in order to investigate their origin. The yeast Saccharomyces cerevisiae was chosen as the model system as it was both simple and relevant. It is a single cellular organism that is genetically manipulable and has a completed genome that is compact with relatively few introns and low amounts of repetitive DNA. However it is also eukaryotic with over 2700 genes having human homologs. A small protein profiling technique was developed using multi-dimensional chromatography. The MC2 strain of yeast was used to reduce in vitro proteolysis as it was deficient in several proteolytic enzymes. Several sample preparation techniques were compared including both selective and non-selective extractions. A pool of small proteins was obtained using size exclusion chromatography under denaturing conditions and the pool resolved by a mixture of high performance ion exchange and reverse phase chromatography. Protein profiles were obtained by either UV absorbance at 215 nm, MALDI-TOF or in line ESI-TOF mass spectrometry. Protein identification was performed by either Edman sequencing of purified species or by tandem mass spectrometry in conjunction with a custom database of every potential ORF in S. cerevisiae. It was found that small proteins constitute only a minor amount of the cellular proteins and that the ribosomal sub-unit family of proteins dominates this population. Initial attempts to profile small proteins by ESI-TOF mass spectrometry indicated that there were far more small proteins in the cell than would be accounted for by the databases. It was found that it was not feasible to purify proteins sufficiently for sequence analysis except for high abundant species. Therefore alternative identification methods were investigated. Tandem mass spectrometry was able to identify small proteins from tryptic digests of protein mixtures and initial experiments identified 61 proteins. This included products of three genes that had only been annotated in the last 4 years demonstrating the suitability of this method for identifying novel small genes from their protein products.
Notlar:
School code: 1543
Tüzel Kişi Ek Girişi:
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(684312.1) | 684312-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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