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The formation of the labyrinth layer is a critical step of placental development. The transcription factor glial cells missing 1 (Gcm1) plays a pivotal role in labyrinth development, but the sequence of events controlling its expression has not been identified yet. Our studies presented herein show that Gcm1 expression occurs in three distinct phases during placental development, each specific to a particular stage of chorio-allantois interaction. In the first, the pre-fusion phase, Gcm1 mRNA is expressed in isolated clusters of chorionic cells, but not efficiently translated. Upon allantois–chorion fusion, the second phase, Gcm1 expression is greatly induced in clusters of chorionic cells separated by non-expressing cells and the Gcm1 mRNA is translated to protein. In the third phase, the labyrinth formation, cells expressing Gcm1 proliferate, involute in the chorionic plate and branched villi formation begins.

Neurofilaments are central determinants of the diameter of myelinated axons. It is less clear whether neurofilaments serve other functional roles such as maintaining the structural integrity of axons over time. Here we show that an age-dependent axonal atrophy develops in the lumbar ventral roots of mice with a null mutation in the mid-sized neurofilament subunit (NF-M) but not in animals with a null mutation in the heavy neurofilament subunit (NF-H). Mice with null mutations in both genes develop atrophy in ventral and dorsal roots as well as a hind limb paralysis with aging. The atrophic process is not accompanied by significant axonal loss or anterior horn cell pathology. In the NF-M–null mutant atrophic ventral root, axons show an age-related depletion of neurofilaments and an increased ratio of microtubules/neurofilaments. By contrast, the preserved dorsal root axons of NF-M–null mutant animals do not show a similar depletion of neurofilaments. Thus, the lack of an NF-M subunit renders some axons selectively vulnerable to an age-dependent atrophic process. These studies argue that neurofilaments are necessary for the structural maintenance of some populations of axons during aging and that the NF-M subunit is especially critical.

Neurofilaments (NFs) are prominent components of large myelinated axons. Previous studies have suggested that NF number as well as the phosphorylation state of the COOH-terminal tail of the heavy neurofilament (NF-H) subunit are major determinants of axonal caliber. We created NF-H knockout mice to assess the contribution of NF-H to the development of axon size as well as its effect on the amounts of low and mid-sized NF subunits (NF-L and NF-M respectively). Surprisingly, we found that NF-L levels were reduced only slightly whereas NF-M and tubulin proteins were unchanged in NF-H–null mice. However, the calibers of both large and small diameter myelinated axons were diminished in NF-H–null mice despite the fact that these mice showed only a slight decrease in NF density and that filaments in the mutant were most frequently spaced at the same interfilament distance found in control. Significantly, large diameter axons failed to develop in both the central and peripheral nervous systems. These results demonstrate directly that unlike losing the NF-L or NF-M subunits, loss of NF-H has only a slight effect on NF number in axons. Yet NF-H plays a major role in the development of large diameter axons.

Transgenic mice of line MβP3 express bacterial β-galactosidase in oligodendrocytes but not other cells of the CNS. The marker enzyme, demonstrated histochemically or by immunostaining in oligodendrocyte cell bodies and along myelin internodes, appears at the time of myelination and persists thereafter; in transplantation experiments, the marker may serve to indicate both the source of particular cells and their state of differentiation. The subventricular zone of the lateral ventricle, grafted from transgenic to wild-type perinatal recipient mice, yields histochemically labeled oligodendrocytes in surrounding host tissue. When grafts are placed in cerebral cortex near callosal radiations, graft-derived oligodendrocytes are found in cerebral cortex and subcortical white matter as far as 1.5 mm from the site of implant but not in nearby caudoputamen. This study is the first to document differentiation of transplant-derived oligodendrocytes in normal developing CNS. Our results are consistent with the well- established notion that oligodendrocyte progenitors migrate during normal development and suggest that such migration might be guided or restricted by mechanisms yet to be identified.

The human neurofilament (H) promoter contains multiple binding sites for nuclear proteins including a Proximal (Prox) site centered around the sequence GGTTGGACC and an adjacent pyrimidine (Pyr) tract site centered around the sequence CCCTCCTCCCC. Surprisingly binding to a probe containing the Prox/Pyr region of the NF(H) promoter was competed in gel shifts by an oligonucleotide containing only an Sp1 binding site (GGGGCGGGG). Supershift assays with a polyclonal anti-Sp1 antisera confirmed that Sp1 was part of the complex formed with the Prox/Pyr probe. However neither bacterially expressed Sp1 516C or vaccinia virus expressed full-length Sp1 778C bound to the Prox or Pyr sequences in DNase I footprints or gel shift assays. Gel shift competitions and supershift assays with probes containing either Prox or Pyr tract sites alone demonstrated targeting of Sp1 to the Prox binding site and identified a non-Sp1 containing complex which contains a Prox binding protein. Adding exogenous Sp1 to a HeLa nuclear extract enhanced the Sp1-containing complex but had no effect on the Prox complex. These studies show that Sp1 can be targeted to a non-Sp1 site in the human NF(H) promoter through protein/protein interactions with a distinct sequence specific DNA-binding protein.

Brief ectopic expression of Gcm1 in mouse embryonic tail bud profoundly affects the development of the nervous system. All mice from 5 independently derived transgenic lines exhibited either one or both of two types of congenital spinal cord pathologies: failure of the neural tube to close (spina bifida) and multiple neural tubes (diastematomyelia). Because the transgene is expressed only in a restricted caudal region and only for a brief interval (E8.5 to E13.5), there was no evidence of embryonic lethality. The dysraphisms develop during the period and within the zone of transgene expression. We present evidence that these dysraphisms result from an inhibition of neuropore closure and a stimulation of secondary neurulation. After transgene expression ceases, the spinal dysraphisms are progressively resolved and the neonatal animals, while showing signs of scarring and tissue resorption, have a closed vertebral column. The multiple spinal cords remain but are enclosed in a single spinal column as in the human diastematomyelia. The animals live a normal life time, are fertile and do not exhibit any obvious weakness or motor disabilities.

We used the yeast two-hybrid assay to identify novel proteins that interact with the D1dopamine receptor. The third cytoplasmic loop (residues 217–273) of the rat D1receptor was used as bait to identify clones encoding interacting proteins from a rat brain cDNA library. This identified two clones encoding the C terminus of rat neurofilament-M (NF-M) (residues 782–846). The NF-M clone did not interact with the third cytoplasmic loops of the rat D2, D3, or D4receptors, but showed weak interaction with that of the D5receptor. Coexpression of full-length NF-M with the D1receptor in HEK-293 cells resulted in >50% reduction of receptor binding accompanied by a reduction in D1receptor-mediated cAMP accumulation. NF-M had no effect on the expression of other dopamine receptor subtypes. Using a D1receptor-green fluorescent protein chimera and confocal fluorescence microscopy, we found that NF-M reduced D1receptor expression at the cell surface and promoted accumulation of the receptor in the cytosol. Interestingly, the D1receptors that were expressed at the cell surface in the presence of NF-M were resistant to agonist-induced desensitization. Cellular colocalization of NF-M and the D1receptor in the rat brain was examined by epifluorescence microscopy. These experiments showed that ∼50% of medium-sized striatal neurons expressed both proteins. Colocalization was also observed in pyramidal cells and interneurons within the frontal cortex. Similar immunohistochemical analyses using NF-M-deficient mice showed decrements in D1receptor expression compared with control mice. These results suggest that NF-M interacts with the D1receptorin vivoand may modify its expression and regulation.

Although gcm was first recognized for its role in specifying glial cell fate in Drosophila melanogaster, its mammalian counterparts are expressed predominantly in non-neural tissues. Here we demonstrate expression of the mouse and human GCM 1 proteins in placenta. We have prepared a highly specific antibody that recognizes the GCM 1 protein and have used it to assess the temporal and spatial expression profile of the protein. In both mouse and human placenta, the protein is associated with cells that are involved with exchange between maternal and fetal blood supplies: the labyrinthine cells of the mouse placenta and the syncytio- and cytotrophoblasts of the human placenta. Using the full-length h Gcm 1 cDNA as a probe, we have mapped the gene on human chromosome 6p12 by fluorescent in situ hybridization.

Oligodendrocyte-specific protein (OSP)/claudin-11 is a recently identified transmembrane protein found in CNS myelin and testis with unknown function. Herein we demonstrate that Osp null mice exhibit both neurological and reproductive deficits: CNS nerve conduction is slowed, hindlimb weakness is conspicuous, and males are sterile. Freeze fracture reveals that tight junction intramembranous strands are absent in CNS myelin and between Sertoli cells of mutant mice. Our results demonstrate that OSP is the mediator of parallel-array tight junction strands and distinguishes this protein from other intrinsic membrane proteins in tight junctions. These novel results provide direct evidence of the pivotal role of the claudin family in generating the paracellular physical barrier of tight junctions necessary for spermatogenesis and normal CNS function.

Neurofilaments (NFs) are prominent components of large myelinated axons and probably the most abundant of neuronal intermediate filament proteins. Here we show that mice with a null mutation in the mid-sized NF (NF-M) subunit have dramatically decreased levels of light NF (NF-L) and increased levels of heavy NF (NF-H). The calibers of both large and small diameter axons in the central and peripheral nervous systems are diminished. Axons of mutant animals contain fewer neurofilaments and increased numbers of microtubules. Yet the mice lack any overt behavioral phenotype or gross structural defects in the nervous system. These studies suggest that the NF-M subunit is a major regulator of the level of NF-L and that its presence is required to achieve maximal axonal diameter in all size classes of myelinated axons.

Previous studies have shown that rodent neurofilaments (NF) are obligate heteropolymers requiring NF-L plus either NF-M or NF-H for filament formation. We have assessed the competence of human NF-L and NF-M to assemble and find that unlike rat NF-L, human NF-L is capable of self-assembly. However, human NF-M cannot form homopolymers and requires the presence of NF-L for incorporation into filaments. To investigate the stage at which filament formation is blocked, the rod domains or the full-length subunits of human NF-L, human NF-M, and rodent NF-L were analyzed in the yeast "interaction trap" system. These studies demonstrated that the fundamental block to filament formation in those neurofilaments that do not form homopolymers is at the level of dimer formation. Based on theoretical biophysical considerations of the requirements for the formation of coiled-coil structures, we predicted which amino acid differences were likely to be responsible for the differing dimerization potentials of the rat and human NF-L rod domains. We tested these predictions using site-specific mutagenesis. Interestingly, single amino acid changes in the rod domains designed to restore or eliminate the coiled-coil propensity were found respectively to convert rat NF-L into a subunit capable of homopolymerization and human NF-L into a protein that is no longer able to self-assemble. Our results additionally suggest that the functional properties of the L12 linker region of human NF-L, generally thought to assume an extended beta-sheet conformation, are consonant with an alpha-helix that positions the heptad repeats before and after it in an orientation that allows coiled-coil dimerization. These studies reveal an important difference between the assembly properties of the human and rodent NF-L subunits possibly suggesting that the initiating events in neurofilament assembly may differ in the two species.

Pelizaeus-Merzbacher disease (PMD) is a dysmyelinating disease resulting from mutations, deletions, or duplications of the proteolipid protein (PLP) gene. Distinguishing features of PMD include pleiotropy and a range of disease severities among patients. Previously, we demonstrated that, when expressed in transfected fibroblasts, many naturally occurring mutant PLP alleles encode proteins that accumulate in the endoplasmic reticulum and are not transported to the cell surface. In the present communication, we show that oligodendrocytes in an animal model of PMD, the msd mouse, accumulate Plp gene products in the perinuclear region and are unable to transport them to the cell surface. Another important aspect of disease in msd mice is oligodendrocyte cell death, which is increased by two- to threefold. We demonstrate in msd mice that this death occurs by apoptosis and show that at the time oligodendrocytes die, they have differentiated, extended processes that frequently contact axons and are expressing myelin structural proteins. Finally, we define a hypothesis that accounts for pathogenesis in most PMD patients and animal models of this disease and, moreover, can be used to develop potential therapeutic strategies for ameliorating the disease phenotype.

qkI, a newly cloned gene lying immediately proximal to the deletion in thequakingviablemutation, is transcribed into three messages of 5, 6, and 7 kb. Antibodies raised to the unique carboxy peptides of the resulting QKI proteins reveal that, in the nervous system, all three QKI proteins are expressed strongly in myelin-forming cells and also in astrocytes. Interestingly, individual isoforms show distinct intracellular distributions: QKI-6 and QKI-7 are localized to perikaryal cytoplasm, whereas QKI-5 invariably is restricted to the nucleus, consistent with the predicted role of QKI as an RNA-binding protein. Inquakingviablemutants, which display severe dysmyelination, QKI-6 and QKI-7 are absent exclusively from myelin-forming cells. By contrast, QKI-5 is absent only in oligodendrocytes of severely affected tracts. These observations implicate QKI proteins as regulators of myelination and reveal key insights into the mechanisms of dysmyelination in thequakingviablemutant.

Alterations in neurofilaments are a common occurrence in neurons of the human nervous system during aging and diseases associated with aging. Such pathologic changes may be attributed to species-specific properties of human neurofilaments as well as cell-type-specific regulation of this element of the cytoskeleton. The development of transgenic animals containing human neurofilament subunits offers an opportunity to study the effects of aging and other experimental conditions on the human-specific form of these proteins in a rodent model. The present study shows that mice from the transgenic line NF(M)27, which express the human midsized neurofilament subunit at low levels (2–25% of the endogenous NF-M), develop neurofilamentous accumulations in specific subgroups of neurons that are age dependent, affecting 78% of transgenic mice over 12 months of age. Similar accumulations do not occur in age-matched, wild-type littermates or in 3-month-old transgenic mice. In 12-month-old transgenic mice, somatic neurofilament accumulations resembling neurofibrillary tangles were present predominantly in layers III and V of the neocortex, as well as in select subpopulations of subcortical neurons. Intraperikaryal, spherical neurofilamentous accumulations were particularly abundant in cell bodies in layer II of the neocortex, and neurofilament-containing distentions of Purkinje cell proximal axons occurred in the cerebellum. These pathological accumulations contained mouse as well as human NF subunits, but could be distinguished by their content of phosphorylation-dependent NF epitopes. These cytoskeletal alterations closely resemble the cell-type-specific alterations in neurofilaments that occur during normal human aging and in diseases associated with aging, indicating that these transgenic animals may serve as models of some aspects of the pathologic features of human neurodegenerative diseases.

The induction of autoantibodies and their possible role in the pathogenesis of autoimmune disease are poorly understood. Involvement of infectious agents has been suspected, but direct evidence is sparse. Whether immunological unresponsiveness to self by antibody-forming B cells is maintained by clonal abortion, clonal anergy or suppression, or how the scenario of interactions between helper T cells, B cells and antigen-presenting cells is distorted in autoantibody responses, is being analysed and widely debated. To evaluate tolerance of neutralizing B-cell responses we used transgenic mice expressing the cell membrane associated glycoprotein (G) of vesicular stomatitis virus (VSV) as self-antigen. We show that autoantibodies to VSV-G cannot be induced by VSV-G in adjuvant or by recombinant vaccinia virus expressing VSV-G, but are triggered by infection with wild-type VSV. The data show that helper T-cell tolerance is crucial in maintenance of B-cell non-reactivity and that cognate T-B recognition is necessary to break tolerance of self-reactive B cells. These results may help to understand mechanisms of virus-induced autoimmunity.

The 3′ termini of RNA from vesicular stomatitis virus and from three widely dissimilar defective interfering particles of the virus are PypGpU-OH. The possible relevance of these findings to autointerference and to replication of vesicular stomatitis virus is discussed.

The base sequence at the 3' end of vesicular stomatitis virus RNA was determined by using terminal labels and chemical RNA sequencing. The leader RNA was complementary to 47 bases at the 3' terminus, whereas the nucleocapsid gene (N) began 51 nucleotides from the 3' end of the genomic RNA. The intervening bases were 3'...GAAA...5' for the Indiana serotype and 3'...GAAAA...5' for the New Jersey serotype. The complements of these bases did not appear in either the leader RNA or the N mRNA. This sequence may function as a stop signal or cleavage site during transcription. Furthermore, processing or termination at this sequence must be inhibited during the production of full-length RNA plus-sense strands (replication). We recently found similar sequences approximately 46 to 48 nucleotides from the 3' ends of several defective interfering particle RNAs where the short defective interfering particle transciption products terminate. This sequence is present also at the end of the polymerase (L) gene.

We have isolated cDNA clones encoding the four different forms of mouse myelin basic protein (MBP) and have analyzed the structure of the MBP gene. The three larger forms of MBP differ from the smallest by the inclusion of either or both of two short amino acid sequences at positions 57 and 124 of the smallest protein. The mouse genome contains a single MBP gene comprised of seven exons. The two amino acid sequences present only in the larger MBPs are encoded by separate exons. Furthermore, all exons in the coding region begin or end in complete codons so that alternative splicing does not alter the reading frame. We conclude that the four forms of this myelin protein are encoded in separate mRNAs, each derived by a simple alternative splicing of the primary MBP gene transcript. Comparison of the amino acid sequence encoded by each exon with a recent model of the secondary structure of MBP suggests that each of the seven exons encodes one or two of the predicted structural motifs of the protein.

The 5'-terminal nucleotide sequence from positions 50 to 130 of vesicular stomatitis virus RNA was determined indirectly by using a defective interfering particle RNA which contains covalently linked genomic minus and antigenomic plus sense RNAs. The last 18 nucleotides of the L gene coding for in the viral polymerase were identified and isolated by specific duplex formation between 5' terminally labeled oligonucleotides from a small single-stranded defective interfering particle RNA and L gene mRNA. The L gene ends at position 60 from the 5' terminus of the vesicular stomatitis genome. The data demonstrated that the first seven adenine residues in the polyadenylic acid tail of L gene mRNA may be coded for in the genome and suggested that the viral transcriptase itself may carry out polyadenylation, possibly by chattering at the uridine-rich sequence at the end of the L gene. Analysis of the 5'-terminal sequence of vesicular stomatitis virus genomic RNA revealed that it might fold into a complex secondary structure with possibly 62% of the bases paired.

Two temperature-sensitive (ts) mutants of the M protein of vesicular stomatitis virus (tsG31 and tsG33) are defective in viral assembly, but the exact nature of this defect is not known. When infected cells are switched from nonpermissive (40 degrees C) to permissive (32 degrees C) temperatures in the presence of cycloheximide, tsG33 virus release increased by 100-fold, whereas tsG31 release increased only by 10-fold. Thus, the tsG33 defect is more reversible than that of tsG31. Therefore, we investigated how the altered synthesis and cellular distribution of tsG33 M protein correlates with the viral assembly defect. At 32 degrees C tsG33 M protein is stained diffusely in the cell cytoplasm and later at the budding sites. In contrast, at 40 degrees C the mutant M protein formed unusual aggregates mostly located in the perinuclear regions of virus-infected cells and partially colocalized with G protein in this region. In temperature shift-down experiments, M can be disaggregated and used to some extent for nucleocapsid coiling and budding, which correlates with the virus titer increase. M aggregates also formed after shift-up from 32 to 40 degrees C, indicating a complete dependence of M aggregation on the temperature. Biochemical analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed that at 40 degrees C M protein is detected exclusively in pellet fractions (nuclear and cytoskeleton components), whereas at 32 degrees C M protein is mainly in the cytoplasmic soluble fractions. Furthermore, when the temperature is raised from 32 to 40 degrees C, the distribution of M protein tends to shift from the soluble to the pellet and cytoskeletal fractions. Electron micrographs of immunoperoxidase-labeled M protein showed that at 40 degrees C M aggregates are often associated with the outer nuclear membranes as well as with vesicular structures. No nucleocapsid coiling was observed in these cells, whereas coiling and budding were seen at 32 degrees C in cells where M protein was partly associated with the plasma membrane. We suggest that the tsG33 M protein mutation may produce a reversible conformational alteration which causes M protein to aggregate at 40 degrees C, therefore inhibiting the proper association of M protein with nucleocapsids and budding membranes.

The nucleotide sequences encoding the matrix (M) proteins of measles virus (MV) and canine distemper virus (CDV) were determined from cDNA clones containing these genes in their entirety. In both cases, single open reading frames specifying basic proteins of 335 amino acid residues were predicted from the nucleotide sequences. Both viral messages were composed of approximately 1,450 nucleotides and contained 400 nucleotides of presumptive noncoding sequences at their respective 3' ends. MV and CDV M-protein-coding regions were 67% homologous at the nucleotide level and 76% homologous at the amino acid level. Only chance homology was observed in the 400-nucleotide trailer sequences. Comparisons of the M protein sequences of MV and CDV with the sequence reported for Sendai virus (B. M. Blumberg, K. Rose, M. G. Simona, L. Roux, C. Giorgi, and D. Kolakofsky, J. Virol. 52:656-663; Y. Hidaka, T. Kanda, K. Iwasaki, A. Nomoto, T. Shioda, and H. Shibuta, Nucleic Acids Res. 12:7965-7973) indicated the greatest homology among these M proteins in the carboxyterminal third of the molecule. Secondary-structure analyses of this shared region indicated a structurally conserved, hydrophobic sequence which possibly interacted with the lipid bilayer.

A cDNA clone containing the entire vesicular stomatitis virus nucleocapsid gene was assembled by fusing portions of two partial clones. When the cDNA clone was inserted into a new general-purpose eucaryotic expression vector and introduced into appropriate host cells, abundant N-protein synthesis ensued. The expressed protein was indistinguishable from authentic N protein produced during vesicular stomatitis virus infections. The recombinant N protein was recognized by a polyclonal antibody and two different monoclonal antibodies and could not be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from authentic N. Our results suggest that the recombinant N protein produced in transfected cells rapidly aggregates into high-molecular-weight complexes in the absence of vesicular stomatitis virus genomic RNA.

In multiple sclerosis, a demyelinating disease of young adults, there is a paucity of myelin repair in the central nervous system (CNS) which is necessary for the restoration of fast saltatory conduction in axons1,2. Consequently, this relapsing disease often causes marked disability. In similar diseases of small rodents, however, remyelination can be quite extensive, as in the demyelinating disease caused by the A59 strain of mouse hepatitis virus (MHV-A59)3,4, a coronavirus of mice. To investigate when and where oligodendrocytes are first triggered to repair CNS myelin in such disease, we have used a complementary DNA probe specific for one major myelin protein gene, myelin basic protein (MBP), which hybridizes with the four forms of MBP messenger RNA in rodents5. Using Northern blot and in situ hybridization techniques, we previously found that MBP mRNA is first detected at about 5 days after birth, peaks at 18 days and progressively decreases to 25% of the peak levels in the adult5–7. We now report that in spinal cord sections of adult animals with active demyelination and inflammatory cells, in situ hybridization reveals a dramatic increase in probe binding to MBP-specific mRNA at 2–3 weeks after virus inoculation and before remyelination can be detected by morphological methods. This increase of MBP-specific mRNA is found at the edge of the demyelinating area and extends into surrounding areas of normal-appearing white matter. Thus, in situ hybridization with myelin-specific probes appears to be a useful method for detecting the timing, intensity and location of myelin protein gene reactivation preceding remyelination. This method could be used to elucidate whether such a reactivation occurs in multiple sclerosis brain tissue. Our results suggest that in mice, glial cells react to a demyelinating process with widespread MBP mRNA synthesis which may be triggered by a diffusible factor released in the demyelinated areas.

The developmental program controlling the expression of myelin basic protein (MBP) gene was studied in the rat using the technique of in situ hybridization. A 35S-labeled cDNA clone of mouse MBP encoding an amino acid sequence present in all four of the major forms of rodent MBP was used. The probe hybridized to the tracts of white matter with different intensities, depending on the age of the animal and the region of the brain examined. In the medulla oblongata, maximal hybridization was found in 5- and 7-day-old rats and was confined to the tectospinal tracts, fibers of the seventh cranial nerve, and the spinocerebellar tracts. By 12 days the amount of MBP mRNA had decreased in these areas. In the cerebrum, the greatest amount of MBP mRNA was observed in 17-day-old rats in the radiations of the corpus callosum. Thereafter, the levels decreased but could still be observed in the adult animals. Thus, using this technique, we have been able to demonstrate that the level of MBP-specific mRNA correlates closely with the development of myelin in different regions of the brain.

cDNA clones encoding proteolipid protein (PLP) were isolated from a mouse brain library and sequenced. We describe two transcripts arising from the PLP locus by alternative splicing: the major one encodes the 277-amino acid PLP protein and the minor one corresponds to the DM-20 protein, a PLP-like protein of 20,000 Mr that shares both amino and carboxyl regions with PLP. These two transcripts lack approximately 70 bases in PLP mRNA from the dysmyelinating jimpy mutant. The deletion spans amino acids 208-232; however, this region is present in the jimpy PLP-encoding gene. We propose that the jimpy mutant suffers a point mutation or the deletion of a few bases in the PLP gene that alters the normal splicing pattern and generates partially deleted PLP transcripts.

Oligodendrocytes, the myelin-forming cells of the central nervous system, were cultured from newborn rat brain and optic nerve to allow us to analyze whether two transmembranous myelin proteins, myelin-associated glycoprotein (MAG) and proteolipid protein (PLP), were expressed together with myelin basic protein (MBP) in defined medium with low serum and in the absence of neurons. Using double label immunofluorescence, we investigated when and where these three myelin proteins appeared in cells expressing galactocerebroside (GC), a specific marker for the oligodendrocyte membrane. We found that a proportion of oligodendrocytes derived from brain and optic nerve invariably express MBP, MAG, and PLP about a week after the emergence of GC, which occurs around birth. In brain-derived oligodendrocytes, MBP and MAG first emerge between the fifth and the seventh day after birth, followed by PLP 1 to 2 d later. All three proteins were confined to the cell body at that time, although an extensive network of GC positive processes had already developed. Each protein shows a specific cytoplasmic localization: diffuse for MBP, mostly perinuclear for MAG, and particulate for PLP. Interestingly, MAG, which may be involved in glial-axon interactions, is the first myelin protein detected in the processes at approximately 10 d after birth. MBP and PLP are only seen in these locations after 15 d. All GC-positive cells express the three myelin proteins by day 19. Simultaneously, numerous membrane and myelin whorls accumulate along the oligodendrocyte surface. The sequential emergence, cytoplasmic location, and peak of expression of these three myelin proteins in vitro follow a pattern similar to that described in vivo and, therefore, are independent of continuous neuronal influences. Such cultures provide a convenient system to study factors regulating expression of myelin proteins.

Proteolipid protein (PLP) is the most abundant transmembrane protein in myelin of the central nervous system. Conflicting models of PLP topology have been generated by computer predictions based on its primary sequence and experiments with purified myelin. We have examined the initial events in myelin synthesis, including the insertion and orientation of PLP in the plasma membrane, in rat oligodendrocytes which express PLP and the other myelin-specific proteins when cultured without neurons (Dubois-Dalcq, M., T. Behar, L. Hudson, and R. A. Lazzarini. 1986. J. Cell Biol. 102:384-392). These cells, identified by the presence of surface galactocerebroside, the major myelin glycolipid, were stained with six anti-peptide antibodies directed against hydrophilic or short hydrophobic sequences of PLP. Five of these anti-peptide antibodies specifically stained living oligodendrocytes. Staining was only seen approximately 10 d after PLP was first detected in the cytoplasm of fixed and permeabilized cells, suggesting that PLP is slowly transported from the RER to the cell surface. The presence of PLP domains on the extracellular surface was also confirmed by cleavage of such domains with proteases and by antibody-dependent complement-mediated lysis of living oligodendrocytes. Our results indicate that PLP has only two transmembrane domains and that the great majority of the protein, including its amino and carboxy termini, is located on the extracellular face of the oligodendrocyte plasma membrane. This disposition of the PLP molecule suggests that homophilic interactions between PLP molecules of apposed extracellular faces may mediate compaction of adjacent bilayers in the myelin sheath.

Ribosomes are observed intimately associated with nucleocapsids of vesicular, stomatitis virus, especially those that line structures that are either cytoplasmic vesicles or invaginations of the plasma membrane.

Summary The kinetics of synthesis and the metabolic stability of uncapped vesicular stomatitis virus (VSV) mRNA transcripts have been studied using techniques which clearly differentiate them from other RNA species. The triphosphate-initiating mRNA transcripts accumulate for at least 5 h during a typical in vitro transcription reaction. The great majority of these transcripts are smaller than a functional message and have been released from the template-transcriptase complex. Label that accumulates in them is stable and is not detectably diminished after a 1 h chase with unlabelled precursor. These kinetic properties are not those expected for active intermediates in mRNA synthesis and suggest that the uncapped transcripts are products of aborted transcription that accumulate during the transcriptive process. However, we cannot rule out that a small subset of these transcripts may be elongated into mature mRNA. Initiation of transcription at internal positions along the VSV genome is both frequent (one-half to one-sixth as frequent as initiations at the leader RNA gene) and site-specific (occurring only at the beginning of cistrons). The relevance of these results to the models for VSV transcription is discussed.

A high degree of conservation exists between the Hox 1.3 homeobox genes of mice and humans. The two genes occupy the same relative positions in their respective Hox 1 gene clusters, they show extensive sequence similarities in their coding and noncoding portions, and both are transcribed into multiple transcripts of similar sizes. The predicted human Hox 1.3 protein differs from its murine counterpart in only 7 of 270 amino acids. The sequence similarity in the 250 base pairs upstream of the initiation codon is 98%, the similarity between the two introns, both 960 base pairs long, is 72%, and the similarity in the 3' noncoding region from termination codon to polyadenylation signal is 90%. Both mouse and human Hox 1.3 introns contain a sequence with homology to a mating-type-controlled cis element of the yeast Ty1 transposon. DNA-binding studies with a recombinant mouse Hox 1.3 protein identified two binding sites in the intron, both of which were within the region of shared homology with this Ty1 cis element.

The nucleotide sequences at the 5' and 3' termini of RNA isolated from the New Jersey serotype of vesicular stomatitis virus [vsV(NJ)] and two of its defective interfering (DI) particles have been determined. The sequence differs from that previously demonstrated for the RNA from the Indiana serotype of VSV at only 1 of the first 17 positions from the 3' terminus and at only 2 of the first 17 positions from the 5' terminus. The 5'-terminal sequence of VSV(NJ) RNA is the complement of the 3'-terminal sequence, and duplexes which are 20 bases long and contain the 3' and 5' termini have been isolated from this RNA. The RNAs isolated from DI particles of VSV(NJ) have the same base sequences as do the RNAs from the parental virus. These results are in sharp contrast to those obtained with the Indiana serotype of VSV and its DI particles, in which the 3'-terminal sequences differ in 3 positions within the first 17. However, with both serotypes, the 3'-terminal sequence of the DI RNA is the complement of the 5'-terminal sequence of the RNA from the infectious virus. These findings suggest that the 3' and 5' RNA termini are highly conserved in both serotypes and that the 3' terminus of DI RNA is ultimately derived by copying the 5' end of the VSV genome, as recently proposed (D. Kolakofsky, M. Leppert, and L. Kort, in B. W. J. Mahy and R. D. Barry, ed., Negative-Strand Virus and the Host Cell, 1977; M. Leppert, L. Kort, and D. Kolakofsky, Cell 12:539-552, 1977; A. S. Huang, Bacteriol. Rev. 41:811-8218 1977).

We have examined the intracellular levels of the guanosine tetraphosphate, guanosine 5'-diphosphate 2'- or 3'-diphosphate (ppGpp), in an isogenic pair of stringent and relaxed strains of Escherichia coli. As previously observed, relaxed strains do not accumulate ppGpp during amino acid deprivation, while stringent strains do. However, under all other culture conditions examined, relaxed and stringent cells regulate their ppGpp levels similarly. During carbon deprivation or step-down transitions both strains accumulate ppGpp. Neither strain accumulates appreciable amounts during phosphate deprivation or after treatment with colicin E1. The basal levels of ppGpp observed in both stringent and relaxed strains during balanced growth are, in most cases, the same, and vary inversely with the growth rate and RNA content of the cells.

The rates of synthesis and chain elongation of RNA in stringent (CP 78) and relaxed (CP 79) strains of Escherichia coli have been measured at 27° in the presence and in the absence of a required amino acid. The following observations have been made. 1. Although the rate of uptake of uracil by CP 78 is reduced 200-fold by amino acid starvation, the rate of synthesis of RNA obtained from the specific activity of the intracellular UTP pool is reduced only 9-fold. The rate of synthesis of RNA in CP 79 is unaffected by amino acid deprivation. 2. The number of nascent RNA chains is approximately the same for CP 78 and CP 79, and is unchanged by amino acid starvation. 3. The rates of RNA chain elongation are 23 and 3 nucleotides per sec for unstarved and starved cultures of CP 78, respectively. The rate for CP 79 is virtually unchanged by starvation: 24 and 21 nucleotides per sec for unstarved and starved cultures, respectively. 4. The results are inconsistent with a purely "noncoordinate" mode of RNA regulation, and suggest that the synthesis of both stable RNA and messenger RNA is restricted, although the relative degree of restriction on the synthetic rates of the two classes cannot be evaluated from these data.

The synthesis of RNA in two strains of Escherichia coli 15 TAU and CP 78, has been studied. With 3H-uracil-labeling data and the measured specific activity of cellular UTP pools over very short labeling periods at 27°, several observations have been made. 1. The initial observed rate of RNA synthesis is approximately 2.7 times faster than the rate of accumulation of stable RNA. Thus, about 60% of the cellular capacity for RNA synthesis is devoted to production of mRNA. 2. The number of nascent RNA chains in 15 TAU and CP 78 is 5100 chains per cell and 2500 chains per cell, respectively. 3. The two strains of E. coli had different growth rates and different observed rates of RNA synthesis, but the rates of chain elongation were 20 nucleotides per sec per chain for 15 TAU and 23 nucleotides per sec per chain for CP 78. This observation suggests that cellular control of the rate of RNA synthesis operates via regulation of the number of nascent chains, not their rate of elongation. 4. It is estimated that RNA polymerase molecules involved in the synthesis of ribosomal RNA are spaced at approximate intervals of 80 DNA base pairs and that these cistrons must be transcribed tandemly by multiple molecules simultaneously.

Two separate protein factors that markedly stimulate DNA-dependent RNA polymerase have been partially purified from ribosome-free extracts of E. coli Q13 cells. These proteins appear to differ from sigma, M, H, psi, and the factor described by Mahadik and Srinivasan. The stimulation by either factor requires the presence of the sigma protein. Using RNA-DNA hybridization competition analyses, we also demonstrate that neither of the new factors preferentially stimulates the synthesis of ribosomal RNA. The purification, properties, and mechanism of action of these new factors are examined.

The rate of RNA synthesis in a stringent strain of Escherichia coli was measured before and during amino acid deprivation. Upon amino acid deprivation the rate of RNA synthesis rapidly fell to 30% of the control rate and did not significantly change from that value upon prolonged starvation. The composition of RNA synthesized during amino acid deprivation was investigated by hybridization-competition, methyl labeling with 14C-methyl-labeled methionine, and polyacrylamide-agarose gel electrophoresis. Of the RNA pulse labeled during amino acid deprivation, ribosomal and transfer species comprise about 12% of the total, as opposed to about 40% in the unstarved control. Consequently, although some ribosomal and transfer RNAs are synthesized during amino acid starvation, the RNA is considerably enriched in messenger RNA, and the major effect of amino acid deprivation is the preferential curtailment of stable RNA synthesis. Our results also indicate that the rate of messenger RNA synthesis is reduced to 50% during amino acid deprivation.

The presence of the altered lysine transfer ribonucleic acid (tRNA) in Bacillus subtilis spores is strongly dependent on the medium on which the cells were sporulated. Cells sporulated on synthetic media or dilute complex media contain little or none of the new component, whereas those sporulated on concentrated complex media accumulate the altered tRNA. The accumulation begins during the fifth or sixth stage of sporulation, the formation of the tunic, and the appearance of refractility, respectively. Mutants blocked early in sporulation differ in their ability to accumulate the altered tRNA when cultured on the same complex medium. Of the four mutants examined, one failed to accumulate any of the RNA, whereas a second contained the full complement characteristic of spores. The third and fourth mutant contained small amounts of the material. It is tentatively concluded that the accumulation of the altered lysine tRNA is not obligate to sporulation but is an epiphenomenon of the process.

A DNA-RNA polymerase complex has been isolated from EDTA-treated Escherichia coli MRE 600. This fraction contains virtually all of the RNA synthetic capacity expressed in the EDTA-treated cells. The in vitro synthesis is sensitive to rifampicin, indicating that the complex is actively initiating RNA chains. Analysis of the product RNA by hybridization competition shows that between 20 to 30% is rRNA. Guanosine tetraphosphate (ppGpp) inhibits global RNA and rRNA synthesis equally, and no preferential inhibition of rRNA synthesis is detected.

Viral assembly was studied by viewing platinum replicas of cytoplasmic and outer plasma membrane surfaces of baby hamster kidney cells infected with vesicular stomatitis virus. Replicas of the cytoplasmic surface of the basilar plasma membrane revealed nucleocapsids forming bullet-shaped tight helical coils. The apex of each viral nose cone was anchored to the membrane and was free of uncoiled nucleocapsid, whereas tortuous nucleocapsid was attached to the base of tightly coiled structures. Using immunoelectron microscopy, we identified the nucleocapsid (N) viral protein as a component of both the tight-coil and tortuous nucleocapsids, whereas the matrix (M) protein was found only on tortuous nucleocapsids. The M protein was not found on the membrane. Using immunoreagents specific for the viral glycoprotein (G protein), we found that the amount of G protein per virion varied. The G protein was consistently localized at the apex of viral buds, whereas the density of G protein on the shaft was equivalent to that in the surrounding membrane. These observations suggest that G-protein interaction with the nucleocapsid via its cytoplasmic domain may be necessary for the initiation of viral assembly. Once contact is established, nucleocapsid coiling proceeds with nose cone formation followed by formation of the helical cylinder. M protein may function to induce a nucleocapsid conformation favorable for coiling or may cross-link adjacent turns in the tight coil or both.

Summary The phosphoprotein (NS) gene from the Indiana serotype of vesicular stomatitis virus (VSV; Mudd-Summers strain) was cloned and sequenced. The NS gene encodes a protein of 265 amino acids which was expressed from a simian virus 40 vector in COS cells. The post-translational modification characteristic of viral NS, the extensive phosphorylation of a cluster of serine and threonine residues, was also evident in recombinant NS protein. The NS gene displays a property common to the phosphoprotein genes of negative-strand RNA viruses: the phosphoprotein mRNA has a second open reading frame (ORF) which could encode a small (7500 mol. wt.) protein. Both measles virus and Sendai virus employ the second ORF of their phosphoprotein gene, and the resultant proteins have an amino acid composition similar to that predicted for the VSV ORF. Comparison of phosphoproteins from different VSV strains revealed two conserved domains that we propose are critical for the function of NS in transcription and replication.

A procedure to enrich for the sequences present at the junction between the linked messages in the polycistronic RNAs symthesized in vitro by vesicular stomatitis virus (VSV) is described. Analyses of these sequences show that they contain a precise transcript of both the intercistronic dinucleotide and the pentanucleotide 5'--C-U-G-U-U--3', common to the 5'-end of all VSV cistrons, covalently linked to the 3'-side of the intervening poly(A). The data strongly suggest that the VSV transcriptase polyadenylylates the mRNAs and can then resume direct and precise transcription of the genome-without reinitiation and without skipping nucleotides.

In vitro transcripts of vesicular stomatitis virus (VSV) were either 5′-terminally labeled by incorporation of [β- 32 P]GTP or were selected on Hg-agarose after incorporation of γ-thio-GTP. Capped RNAs ranged in size from 23 nucleotides, the shortest capped RNA detected, to full-length message size. The 5′-terminal sequences corresponded to those of N message and to a small amount of NS message. Approximately 14% of the capped N gene transcripts were terminated at positions 86 to 90 of the VSV genome, giving rise to specific, 36 to 40-nucleotide-long, capped RNA species. The GTP-initiated RNAs were short with a predominant 28-nucleotide-long RNA species. A minor portion was as large as mRNAs. Nucleotide sequence analyses of the short RNA revealed that it was specifically initiated at positon 91 of the VSV genome, 41 nucleotides within the N cistron. This corresponds exactly to the site where transcription of the 40-nucleotide-long, capped RNA terminated. Initiation with GTP at position 91 occurred at approximately the same frequency as termination of the capped RNA at position 90, suggesting that intracistronic initiation at position 91 may depend upon termination of transcription of the 5′-proximal region and therefore may be sequential. This unique RNA represents the first transcript of VSV which was initiated at an intracistronic site with GTP, and may also represent the first example of a transcript derived from a stop/start mechanism of VSV transcription in vitro. Although initiation occurred frequently at the beginning of the N cistron yielding 11 to 14-nucleotide-long, [β- 32 P]ATP-labeled transcripts (D. F. Pinney and S. U. Emerson, J. Virol. 42: 889-896, 1982), capping of these short RNAs was not detected. This suggests that transcripts may have to be 15 to 23 nucleotides long to be accepted as substrates by the guanyltransferase.

We have isolated cDNA clones encoding three separate forms of human myelin basic protein (MBP), 21.5, 18.5, and 17.2 kDa, and have determined the nucleotide sequence of each. The three forms share a common sequence but differ by the inclusion of a 26-residue amino acid sequence near the N terminus of the 21.5-kDa protein or by the absence of an 11-residue amino acid sequence near the C terminus of the 17.2-kDa protein. The sequences either added to or deleted from the major 18.5-kDa MBP correspond exactly to exons 2 and 5 of the mouse MBP gene, suggesting that the human and mouse genes have similar exon structures. We have also identified the 21.5-kDa human MBP on immunoblots using antisera raised to a peptide encoded by the mouse exon 2 sequence. Southern blotting studies of human genomic DNA reveal a simple pattern consistent with a single human MBP gene. Thus, the three MBP mRNAs are likely to arise from alternative splicing of a primary human MBP transcript. Conservation of the 26 amino acid mouse exon 2 sequence in human MBP suggests an important role for this sequence in myelination.