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A MODEL FOR HISTONE DISTRIBUTION IN MAMMALIAN SPERM CHROMATIN USING PULSE FIELD GEL ELECTROPHORESIS.
Sotolongo, Barbara1, Ward, W. Steven1, 1
ABSTRACT- Chromatin in hamster sperm is uniquely compacted. Most of sperm chromatin is bound by protamines into tightly compacted donut-shaped structures which are DNAse resistant, and each contain an average of 50 kb of DNA. But, a small portion of sperm chromatin remains bound to histones into DNAse sensitive nucleosomes, as in somatic cells. Here, we test a model that the histone bound component of sperm chromatin is a"linker" chromatin between adjacent protamine-donuts. The use of pulse field gel electrophoresis (PFGE) for studying this proposed organization in hamster sperm chromatin was used because it allows separation of large amounts of DNA. We used three experimental conditions to study the histone distribution in sperm nuclei; (1) untreated spermatozoa, (2) Triton X-100 washed, isolated nuclei, and (3) DNAse treated Triton X-100 washed nuclei. Untreated spermatozoa resulted in an elongated compacted zone in the higher migration areas. The results for both isolated nuclei and DNAse I treated sperm nuclei were similar, a smear of DNA with an average size of 50 kb. These preliminary experiments suggest that a DNAse sensitive component exists at 50 kb intervals interspersed throughout the sperm chromatin. They also suggest that sperm nuclei contain an endogenous nuclease that can be activated under appropriate conditions. These data support our model for histone bound DNA being interspersed in between the protamine donuts.
KEY WORDS: histone, sperm, mammalian, Pulse field gel electrophoresis