Jamie Boorech

Defense Date

7-11-2003

Graduation Date

Summer 2003

Availability

Immediate Access

Submission Type

thesis

Degree Name

MS

Department

Biological Sciences

Committee Chair

Richard P. Elinson

Committee Member

John S. Doctor

Committee Member

Nancy Trun

Keywords

Dazl, germ plasm, Rana pipiens

Abstract

The DAZ gene on the human Y chromosome has been implicated in male fertility. Humans and other animals possess an autosomal version of this gene, referred to as DAZ-like or Dazl, which functions in gametogenesis and fertility. In order to expand our knowledge of Dazl, I cloned Dazl from the northern leopard frog Rana pipiens. To obtain the clone, I screened a R. pipiens ovary cDNA library with a RpDazl-specific PCR-generated radioactive probe. RpDazl, as determined by sequencing, is 3.5kb in length, with an 843 nucleotide open reading frame (ORF) and a 3' untranslated region (UTR) of 2635 nucleotides. At the amino acid level, the ORF of RpDazl is 51.8% identical to the ORF of Xenopus laevis Dazl or XDazl and 53.6% identical to the ORF of human DAZL. RpDazl has a characteristic RNA recognition motif (RRM) that is 73% identical to the RRM in X. laevis, and contains two smaller motifs within the RRM, RNP-1 and RNP-2. These two smaller motifs are almost 100% conserved across all species studied. RpDazl also contains a DAZ repeat, another characteristic domain of Dazl genes. The DAZ repeat of RpDazl is 69% identical to the DAZ repeat of XDazl. Northern blot analysis revealed a transcript length of 3.8kb and 3.3kb in two independent trials. RT-PCR was performed using template cDNA, made from R. pipiens ovarian RNA, and 23 sets of primers that spanned the cloned sequence. These data authenticated continuity over greater than 3.25kb of the cDNA. Combining results of the Northern blot and RT-PCR experiments, I conclude that the cloned RpDazl is full length. Preliminary data were also obtained regarding the maternal localization of the RpDazl RNA using RT-PCR as well as a computer based sequence analysis program called RepFind. RT-PCR analysis revealed that RpDazl is localized to the vegetal one-third of the oocyte. RepFind revealed clusters of CAC repeats, which are known localization elements. The significance of these clusters, however, is lower than that of the clusters present in the 3' UTR of XDazl. Further analysis will be required to show more precise RpDazl localization, specifically germ plasm association.

Format

PDF

Language

English

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