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Primary muscle cell cultures consisting of single myocytes and fibroblasts are grown on flexible, optically clear biomembranes. Muscle cell growth, fusion and terminal differentiation are normal. A most effective membrane for these cultures is commercially available Saran Wrap. Muscle cultures on Saran will, once differentiated, contract vigorously and will deform the Saran which is pinned to a Sylgard base. At first, the muscle forms a two-dimensional network which ultimately detaches from the Saran membrane allowing an undergrowth of fibroblasts so that these connective tissue cells completely surround groups of muscle fibers. A three-dimensional network is thus formed, held in place through durable adhesions to stainless steel pins. This three-dimensional, highly contractile network is seen to consist of all three connective tissue compartments seen in vivo, the endomysium, perimysium and epimysium. Finally, this muscle shows advanced levels of maturation in that neonatal and adult isoforms of myosin heavy chain are detected together with high levels of myosin fast light chain 3.

Human fetal muscles at ages 110, 125, and 132 days contain a fetal-specific myosin light chain. This light chain is absent in adult human muscle, copurifies with myosin, and is identified as a slow light chain because it reacts with purified antibody to chicken slow muscle light chains and does not react strongly with antibody to fast myosin light chains. This light chain is synthesized in cultures of fetal muscle along with normal myosin light chains. The presence of a fetal light chain in culture provides a marker for studies of human muscle disease in which it is important to know when or if the muscle makes a transition from embryonic or fetal expression to true adult phenotype.

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Abstract. Nucleoli from unfertilized Urechis eggs, labeled with tritiated RNA precursors, have been isolated for simultaneous autoradiographic localization and biochemical analysis of labeled RNA. The production of the ribosomal RNA precursor (38S) and its first cleavage occur at the fibrillar core region of the nucleolus. The products, predominantly 30S RNA, are then rapidly transported and stored in the granular cortex of the nucleolus. The formation of the nucleolar cortex, therefore, seems to result from an accumulation of partially processed ribosomal RNA with its associated proteins.It is now well documented that the nucleolus is a major site of ribosomal RNA (rRNA) synthesis.'-3 RNA is first made in the fibrillar core of the nucleolus and then moves to the granular cortex.4-8 RNA synthesis begins in the newly formed nucleolus, which is mainly composed of fibrillar components; as synthesis continues, granular components gradually accumulate.7-'2 These observations suggest that the rRNA precursor'-3 that is made in the core region of the nucleolus'3 may also be processed there and that the granular cortex represents an accumulated mass of some partially and/or fully processed species of rRNA with associated proteins. The present report deals with a coordinated cytochemical and biochemical study to test this contention.The unfertilized eggs of Urechis are active in RNA synthesis.14'15 The rate of processing of the rRNA precursor in these eggs is relatively slow.'516 This material is, therefore, suitable for a study of where in the nucleolus such a precursor is made and processed and where the products are stored. We have been able to isolate clean samples of Urechis egg nucleoli in which the core and the cortex regions can be clearly distinguished under a light microscope. Such samples can be used simultaneously for autoradiographic localization and biochemical analysis of labeled RNA.Materials and Methods. The unfertilized eggs of the echiuroid marine worm Urechis caupo were collected, filtered, and washed in sea water.'7 They were then incubated for various times in sea water containing [5-3H]uridine (100 uCi/ml; sp act 28 Ci/mmol) or [methyl-3HILmethionine (100 ,Ci/ml; sp act 1.02 Ci/mmol). In other experiments, eggs were first labeled with tritiated uridine, washed, and incubated further in sea water with added unlabeled uridine (1 mg/ml) and actinomycin D (50 Mg/ml; actinomycin D was a gift from Merck, Sharp and Dohme Research Lab., Pennsylvania). All experiments were done at 18-20'C. Some eggs were fixed in acidic alcohol and others were used to isolate nucleolar and non-nucleolar fractions. Packed eggs were mixed with 968