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

Our previous biochemical analyses revealed that the levels of the minor MBP isoforms 21.5 and 17 kDa are elevated relative to the 14 and 18.5 kDa MBP isoforms in the fraction of isolated myelin of murine CNS that is enriched in interlamellar junctions (or radial component). To substantiate the localization of 21.5 and 17 kDa MBP in the myelin sheath, we used immunoelectron microscopy on thin-sections of mouse optic nerve. Two different polyclonal antibodies were used to distinguish 21.5 and 17 kDa MBP from 14 and 18.5 kDa MBP: Ab-MBP21.5, which was raised against a synthetic peptide corresponding to the exon II amino acid sequence 61-83 of mouse 21.5 kDa MBP (LKQSRSPLPSHARSRPGLCHMYK), and Ab-MBP14, which is immunoreactive to all four isoforms of mouse MBP. Our SDS-PAGE/immunoblotting demonstrated that Ab-MBP21.5, unlike Ab-MBP14, recognized only the 21.5 and 17 kDa MBP isoforms from isolated mouse CNS myelin. Immunolabelling of tissue sections indicated that Ab-MBP14 bound tenfold more to junction-free compact myelin than to radial component, whereas Ab-MBP21.5 bound about equally to the two regions of the myelin sheath. In addition, within the junction-free compact myelin, both antibodies bound nearly three fold more to the major dense line than to the intraperiod line.

The aberrant sex-ratio mutation in D. simulans used for this study is a temperature-sensitive autosomal recessive. Homozygous males raised at 16 degrees C produce about 2% of F1, but those raised at 26 degrees C, have a normal sex ratio. Ultrastructural studies of spermatogenesis have revealed many anomalies in the germ cells of flies raised at 16 degrees C, but the same flies raised at 26 degrees C had few anomalies. The earliest spermatogenic stage with noticeable abnormalities was the primary spermatocyte. In later stages there were pronounced abnormalities in nucleoli, chromatin condensation, nuclear shape, Golgi complex, acrosome, and microtubules. There is asynchronous differentiation of spermatids within a bundle. Some of the abnormalities encountered are disorganization or loss of microtubules of the axoneme, degenerating nebenkern derivatives, and increased numbers of lysosomes, multilamellate bodies, and multivesicular bodies. At the lower temperature, more than half of the sperm within the same bundle were found in different stages of degeneration. Genetic analysis suggests that the sex-ratio gene causes abnormalities and degeneration of most of the Y-bearing sperm. However, counts of abnormal sperm at the ultrastructural level indicate that some X-bearing sperm must also undergo degeneration. These observations show that the sex-ratio gene is of variable penetrance at different temperatures in the primary spermatocyte and of differential penetrance in X- and Y-bearing sperm.

Myelinated CNS tissues from homozygous/hemizygous and heterozygous jimpy rumpshaker jprsh mutant mice were examined to determine the consequences on myelin structure of this mutation in the proteolipid protein (PLP) gene. Polyacrylamide gel electrophoresis and immunoblotting of brain homogenates confirmed that there was a decrease in PLP levels on the B6C3 genetic background onto which this gene was bred. We also observed an increase in level of a protein band that could correspond to the uncharacterized 10‐kDa PLP previously reported in jprsh mice on an Rb(1.3) 1Bnr background. High‐performance TLC and densitometry of lipids from brain homogenate and isolated myelin revealed a decrease in content of cerebrosides and sulfatides. Electron microscopy on optic nerves revealed that normal radial component is retained in jprsh myelin, further substantiating that PLP is not a component of this junctional complex. X‐raydiffraction measurements on unfixed optic nerves showed that the jprsh period is 5–10 Å larger than normal. Moreover, jprsh optic nerve myelin was unstable, as evidenced by a continual increase in the period postdissection. jprsh myelin that was equilibrated at varying pH and ionic strength typically had a larger than normal period under all conditions (both swelling and compacting). Our findings thus demonstrate that the biochemical abnormalities in the jprsh mutant correlate with a wider periodicity and less stable packing of the myelin.