The protamine gene cluster, ie the PRM1>PRM2>TNP2 locus, constitutes a tightly regulated genic domain. At left is a representation of human chromosome 16 showing the relative position of the PRM1>PRM2>TNP2 locus at 16p13.2. Cosmid hP3.1 corresponding to this 40,573 bp region has been sequenced in its entirity. Its sequence and annotations can be accessed through GenBank via NCBI under accesion number U15422. Below is a DNAVIEW graphic of the 40,573 bp sequence of the PRM1>PRM2>TNP2 region prepared by Drs. Jim Nelson, Gautam Singh and Stephen Krawetz and published in the Journal of Biological Chemistry (1994) 269: 31067-31073. The four nucleotides are represented as colored bars, where A=Red, C=Green , G=Blue and T=Yellow. A CpG island lies near the 3' end of the sequence.

 

The corresponding physical locus was defined as the DNase I-sensitive domain. This was elucidated by Susan Wykes. The results of these studies were published in the Journal of Biological Chemistry (1995) 270: 8755-8762.

The protamine genic domain resides within a single DNase I-sensitive region. The DNase I-sensitivity profile for each data point can be displayed by clicking on the picture. The open diamonds indicate potential regions of matrix association, or MARs. These were primarily identified by a computer algorithm (Kramer and Krawetz, 1995), and then comfirmed by a biochemical assay (Kramer and Krawetz, 1996). This domain of DNase I sensitivity remains constant throughout spermatogenesis (Kramer et al., 2000)

Highly condensed regions of chromatin (i.e. heterochromatin) are transcriptionally silent as the DNA is not accessible to the "transcription machinery" (i.e. transcription and transcription associtated factors of the preinitiation complex). DNase I sensitivity, which measures the accessiblity (to nuclease activity) of DNA, provides an assay to measure how, at any one time, regions of chromatin are accessible (ie. potentiated). The figure below outlines the steps necessary to promote transcription. The domain must be Potentiated so that the preinitiation complex can associate with elements at the promoter and enahncer. Once this is accomplished, RNA polymerase (shown in red) Initiates transcription. Once the RNA polymerase has cleared the promoter, Elongation of the RNA transcript proceeds.

 

Transgenic and DNase I-sensitivity analysis has led to the following model of gene selection in the haploid spermatid. (Kramer et al. 1998).

The members of the PRM1>PRM2>TNP2 locus are first potentiated in the late pachytene spermatocyte, transcribed in round spermatids, and their products translated at the elongating spermatid stage of differentiation. The Phosphoglycerate kinase genes (Pgk1 and Pgk2) are another example of genes that are differentiatlly regulated by X-inactivation and potentiation throughtout spermatogenesis. When Pgk1 (on the X chromsome) becomes inactivated during meiosis, Pgk2 (an autosomal, testes specific form) is potentiated and transcribed. DNase I hypersensitive sites (red stars) are marked. These regions are commonly related with regulatory regions.

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