Beyond the Identification of Transcribed Sequences: Functional and Expression Analysis

9th Annual Workshop, October 28-31, 1999

Co-sponsored by the U.S. Department of Energy


BORISSEVITCH

Large scale cloning, sequencing and expression profiling of genes expressed in transcription factor CREM dependant manner during mouse spermatogenesis

Igor Borissevitch, Tim Beissbardth, Andreas Hoerlein and Guenter Schuetz

German Cancer Research Center, Heidelberg, Germany

CREM belongs to CREB (cAMP Responsive Element Binding protein) family of transcription factors. CREMtau, an activator splice isoform of the CREM protein  is highly expressed after meiosis in round spermatides at stages from 1 to 5. According to the high level and time restriction of expression, CREM seems to be the major trigger of the expression at the late stages of spermatogenesis. 

This work represents large scale cloning, sequencing and expression profiling of RNA messages expressed in a CREM dependant manner. We have used gene targeting to selectively eliminate the transcription factor CREM (Nature, 1996, vol.380, pp.162-165). CREM -/- mice display a normal phenotype but males are sterile due to an arrest of spermatogenesis.  Spermatide development is blocked at stages 2-5 and results in the absence of cells of all further stages including spermatozoa. By use of subtractive suppression hybridisation we have cloned messages expressed in wild type but not in a CREM -/- mutant mouse. 12000 clones were analysed by sequencing and hybridisation. Redundancy of this library has been reduced by high density filter hybridisation with the most abundant clones. 950 clusters of sequences were obtained. They represent 79 known mouse genes, 81 homologs to known genes (mostly rat and human)  170 different mouse ESTs, 91 ESTs from other spices, 21 homologs to genomic sequences, 139 novel sequences.

These data compile new extensive information about gene expression during spermatogenesis. In addition, these data provide selection of genes to search for direct CREM target genes (for instance, known CREM target gene ACE presents in our library). Based on our results application may be found for diagnostic and therapeutic intervention in infertile patients with spermatogenetic abnormalities.


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