Identification Of A New Gene And Function Of Mouse DNA Methyltransferase 3a And 3b During Spermatogenesis In Drosophila

During spermatogenesis the histones in the chromatin are replaced by other more basic proteins leading to a more condensed structure of the DNA. The amino acid sequence of such proteins is not as conserved as that of histones. In Drosophila it is unknown which protein can carry out this function. Therefore our aim is to find the respective protein. We used the mouse TP2 (transition protein 2) sequence to search in the D.melanogaster protein bank to find a very basic protein which may have functions similar to the transition proteins. One gene was detected which might represent the equivalent to TP2. We recovered the respective gene. By in situ hybridization we found that the gene is transcribed in testis, but it is not a testis-specific gene as would be expected for a TP2-related gene. The gene was inserted into a bacterial expression vector and the encoded protein was synthesized and recovered. We immunized rabbits with this protein and isolated the antiserum. Immunofluoresce demonstrates that it exists mainly in the nuclei. Its function is not yet known. DNA methylation is a kind of post-replicative modification and it is conserved from prokaryotes to eukaryotes. It is believed that DNA methylation has important functions in gene imprinting, for transposons and in vivo retrovirus silencing as well as the stability of the genome; Abnormal methylation is often concomitant with cancer. DNA methylation is mainly accomplished by DNA methyltransferases and these enzymes are more complexed in eukaryotes compared with their homolog in prokaryotes. In eukaryotes DNA methyltransferases fall into three groups according to their regulatory region. But in Drosophila and other insects the DNA methylation level is very low and only the second group homolog has been found until now. Therefore we have produced transgenic flies with mouse Dnmt3a and Dnmt3b. These enzymes are believed to have de novo CpG methylation activity after replication. The genes were introduced into the fly genome. They were only expressed in testis as we wanted to see if methylation has effects on the spermatogenesis. Our data shows that the methyltransferases have no effect on spermatogenesis and that the CpG methylation level is not changed. By RT-PCR we found that they are both transcribed in testis .These results indicate two possibilities:1. methylation has no effect on the Drosophila spermatogenesis. 2.there maybe exist some mechanism to prevent the expression of extraneous genes in the germ cells which is not so surprised considering the function of germ cells. To prove these speculation we need to do further experiment.