Characterization of human mitochondrial translation initiation factor 2 pseudogene 1 / by Patrick J. Enderle.
| Author/creator | Enderle, Patrick J. author. |
| Other author | Farwell, Mary A., degree supervisor. |
| Other author | East Carolina University. Department of Biology. |
| Format | Theses and dissertations |
| Production | 2001. |
| Description | 79 leaves : illustrations (some color) ; 28 cm |
| Supplemental Content | Access via ScholarShip |
| Subjects |
| Summary | Human Mitochondrial Translation Initiation Factor 2 (MTIF2) is a nuclear-encoded protein functioning in the initiation of mitochondrial translation in human cells. To understand more about this protein, a human genomic library was screened with a cDNA probe, and seven genomic clones for the MTIF2 were isolated. Preliminary sequencing of the clones showed three of them to contain copies of a single, potential pseudogene for MTIF2, which we named MTIF2P1 (Pseudogene 1). Further sequencing of these clones showed the sequence to be a processed pseudogene that lacks intron structure, thus resembling the cDNA structure. However, with respect to the cDNA, the pseudogene exhibits a large 5' truncation as well as numerous point mutations and some deletions of sequence. MTIF2P1, 1587 bp long sequence, contains other distinguishing characteristics of a processed pseudogene, including an AT rich sequence, a 3' poly A tail, and flanking direct repeat sequences as well as a higher incidence of transition mutations versus transversions. Therefore, we conclude that MTIF2P1 is a processed pseudogene of MTIF2. The chromosomal location of MTIF2P1 was determined with two different methods. For Polymerase Chain Reaction, unique primers were designed from the pseudogene sequence and cDNA sequence data to screen a Coriell Somatic Cell Hybrid Panel. The presence or lack of PCR products was analyzed using a 1.8% agarose gel stained with ethidium bromide. In addition. Fluorescence In Situ Hybridization (FISH) was used to determine the pseudogene location. The three clones of the pseudogene were labeled with biotin-14-dUTP, combined into a cocktail probe mixture, and hybridized to metaphase chromosome spreads made from human blood cells. These spreads were then treated with avidin and anti-avidin antibodies and stained with propidium iodide to obtain a fluorescent signal. Both procedures localized MTIF2P1 to Chromosome 1. The specific location, Ipl3-pl2, was obtained by G banding. The localization and the sequence data were confirmed using the BLAST search engine, comparing the data to the first draft of the Human Genome. In an attempt to determine its age, the pseudogene data was aligned with the MTIF2 cDNA along with the bovine MTIF2 cDNA using CLUSTALX. Distance measurements, generated by PAUP, were used to perform a relative rate test, only to conclude that MTIF2P1 appears to be mutating more than its functional counterpart MTIF2. While this finding is to be expected, since processed pseudogenes lack selective constraints, the rates of substitution were significantly different, and thus, the age was not able to be determined with this alignment. However, through inferences made from the somatic cell hybrid screening, we have determined that the pseudogene could have been formed less than 65 million years ago. |
| General note | Presented to the faculty of the Department of Biology. |
| General note | Advisor: Mary A. Farwell |
| Dissertation note | M.S. East Carolina University 2001 |
| Bibliography note | Includes bibliographical references (leaves 75-79). |
| Genre/form | dissertations. |
| Genre/form | Academic theses |
| Genre/form | Academic theses. |
| Genre/form | Thèses et écrits académiques. |
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