Date of Award

6-1-2013

Document Type

Campus Access Thesis

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

Kenneth C Kleene

Second Advisor

Linda Huang

Third Advisor

Alexey Veraksa

Abstract

Spermatogenesis is the process of cell renewal and cytodifferentiation that produces male gametes, spermatozoa. It is divided into three phases during which developing male germ cells replicate in the mitotic cycle, undergo meiosis, producing haploid cells, spermatids, which differentiate into spermatozoa. Early spermatids are transcriptionally active, but chromatin remodeling in late spermatids results in the cessation of transcription. Thus, new proteins synthesized for the final stages of sperm differentiation are translated from mRNAs that are transcribed in early spermatids, stored in a translationally repressed state for up to a week, and translationally activated in late spermatids. My thesis concerns two aspects of translational control: using comparative genomics to identify cis-elements that potentially regulate translation of the spergen-1 mRNA, and creating pET plasmids to express full-length recombinant Y-box proteins, MSY3S and MSY3L.

The spergen-1 protein contains a mitochondrial targeting signal and is localized to the mitochondrial sheath in late spermatids. Translation of the spergen-1 mRNA is developmentally regulated; the mRNA is first detected in early spermatids, and the protein is first detected 6 days later. The conserved N-terminal spergen-1 mitochondrial targeting signal was used as a query to search mammalian genomes in ENSEMBL identifying orthologous genes in 20 species of placental mammals, 3 species of marsupial mammals, and a monotreme. The spergen-1 5'UTRs in all 24 species contain short upstream reading frames, a well-known negative translational control element. The termination codons of the upstream reading frames are close to the spergen-1 AUG initiation codon, a position that correlates with strong translational repression.

The second part of this thesis concerns cloning Y-box proteins, RNA binding proteins, which are thought to repress mRNA translation in early spermatids. Y-box protein 3, MSY3, is expressed as two isoforms differing by 69 amino acids, neither of which has been expressed as a full-length recombinant protein. In this study, cDNAs encoding full-length long and short isoforms were PCR-amplified and cloned into the pET11b vector for expression in E. coli. These clones will be used in the future studies of the specificity of each isoform in mRNA binding and translational repression.

Comments

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