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Development is based on spatially and temporally regulated expression of the genetic information contained in embryonic cells. In Drosophila melanogaster, at least three classes of genes are involved in the regulation of expression of genes for the establishment of the body pattern and segment identity. Among them, the homeotic genes responsible for the segment identity have been shown as being clustered in two major loci: the Antennapedia complex (Ant-C) and the Bithorax complex (BX-C) (Gehring, W., Review, 1985). A highly conserved sequence of 180 bp, the horneo box, has been observed repeatedly in this class of genes. This horneo box encodes a 60 amino acid protein domain, the horneo domain. These horneo box sequences have recently been shown in the genomes of a number of species including mice and humans.

Genomic clones for the largest human neurofilament protein (NF-H) were isolated, the intron/exon boundaries mapped and the entire protein-coding regions (exons) sequenced. The predicted protein contains a central region that obeys the structural criteria identified for alpha-helical 'rod' domains typically present in all IF protein components: it is approximately 310 amino acids long, shares amino acid sequence homology with other IF protein rod domains and displays the characteristic heptad repeats of apolar amino acids which facilitate coiled-coil interaction. Nevertheless, anomalies are noted in the structure of the NF-H rod which could explain observations of its poor homopolymeric assembly in vitro. The protein segment on the carboxy-terminal side of the human NF-H rod is uniquely long (greater than 600 amino acids) compared to other IF proteins and is highly charged (greater than 24% Glu, greater than 25% Lys), rich in proline (greater than 12%) and impoverished in cysteine, methionine and aromatic amino acids. Its most remarkable feature is a repetitive sequence that covers more than half its length and includes the sequence motif, Lys-Ser-Pro (KSP) greater than 40 times. Together with the recent identification of the serine in KSP as the main target for NF-directed protein kinases in vivo, this repetitive character explains the massive phosphorylation of the NF-H subunit that can occur in axons. The human NF-H gene has three introns, two of which interrupt the protein-coding sequence at identical points to introns in the genes for the two smaller NF proteins, NF-M and NF-L.(ABSTRACT TRUNCATED AT 250 WORDS)

Shiverer (shi) is an autosomal recessive mutation in the mouse characterized by an almost total lack of central nervous system myelin. While small amounts of other myelin components are present in the brain of the shi mouse, the four forms of myelin basic protein (MBP) are not detectable. Previous investigations by us and others indicate that the MBP gene has undergone a major rearrangement in the shi mutant. Herein, we report in detail the nature and extent of the rearrangement: a 20-kilobase region within the MBP gene is missing in the mutant. We map the 5' breakpoint of the deletion to the second intron and the 3' breakpoint to a site 2 kilobases beyond the last MBP exon. The junction of the upstream and downstream portions of the gene contains only one nucleotide not accounted for by the wild-type MBP gene sequence. The 3' side of the deletion occurs in the 3rd of 11 tandem repeats of a 31-base-pair sequence. This region is rich in alternating purine and pyrimidine stretches, sequences that have been associated with both Z-DNA structures and gene rearrangements. The recombination junction shares several features with the junctions characterized by Anderson et al. [Anderson, R., Kato, S. & Camerini-Otero, D. (1984) Proc. Natl. Acad. Sci. USA 81, 206-210] in mouse L cells and is consistent with their model for a partially homologous recombination event. The structure of the shi recombination junction suggests that the donor DNA molecules were aligned in a partially homologous region before staggered cutting and joining occurred.

Expression of the murine homeobox containing gene Hox-1.3 was analyzed in mouse embryos using polyclonal antisera to peptides predicted from cDNA and genomic sequences. At the earliest stage examined, 7.5 days gestation, cell nuclei throughout the three embryonic germ layers and in extraembryonic structures derived from the fertilized ovum were strongly immunoreactive. Rostro-caudal gradients or other patterns of regional differentiation in levels of expression could not be seen. Surrounding maternal tissue showed only weak immunoreactivity. At 8.5 days gestation, immunoreactivity was present in all embryonic structures including neural tube, somites and lateral plate mesoderm, ectoderm and endoderm. Immunoreactivity was progressively restricted thereafter. At 17 days gestation, strong immunoreactivity was largely restricted to the nervous system, both central and peripheral. Spinal cord was well stained, with a dramatic reduction in intensity near the junction of spinal cord and brain. In addition to this overall pattern, enhanced immunoreactivity appeared in limited populations of newly-formed neuroblasts of spinal cord and brain, suggesting that Hox-1.3 might serve to regulate the development of specific types of neurons following cessation of precursor cell mitosis.

The human brain contains four isoforms of myelin basic protein (MBP), previously identified by cDNA cloning. We have now isolated and characterized genomic clones encoding the human MBP gene. The gene is 45 kb in extent and consists of seven exons. Alternative splicing of the primary MBP transcript can account for all four human MBP isoforms. The intron-exon boundaries of the gene have also been determined, and all conform to the known consensus splice sequences. These sequences, however, do not explain the alternative splicing pattern found in human brain. Transcription of the human MBP gene begins at a single site within the MBP promoter, and all four MBP isoforms are transcribed from this same site. The promoter region does not contain any known sequence elements, but does have a 12-bp sequence also found in the JC virus 98-bp tandem repeat. A relative gradient of MBP transcription is found from caudal to rostral within the developing human brain, which parallels the known sequence of myelination found in these areas. RNase protection of brain RNA demonstrates more of the 21.5-kD and 20.5-kD MBP mRNAs in neonatal brain than in the adult frontal cortex, which suggests that alternative splicing of the primary MBP transcript is also regulated temporally during myelin development. These data show that regulation of myelination is complex, involving regional cellular interactions and trans activation of transcription, as well as modulation of alternative splicing. Comparison of the human and mouse data also suggests that alternative splicing plays an important role in myelin biogenesis.

The myelin-deficient (MD) rat does not express the major protein of CNS myelin, proteolipid protein (PLP). Here we further analyze whether this defect is reflected at the level of mRNA and whether the expression of other myelin proteins is affected in oligodendrocytes in vivo and in vitro. Both myelin basic protein (MBP) and PLP message levels were reduced in MD rats to 10–20% of the normal littermate controls, while the level of expression of an astrocyte-specific gene, glial fibrillary acidic protein (GFAP), was normal. Although MBP and PLP mRNAs were equally depressed, only MBP was detected with immunolabeling of corpus callosum, while PLP was absent in oligodendrocytes both in vivo and in vitro. A reduced number of MD rat oligodendrocytes express MBP in vitro compared to controls. The MD rat optic nerve contains normal numbers of 0–2A progenitors, but they tend to differentiate into GC-positive oligodendrocytes faster than oligodendrocytes from control littermates. In conclusion, the absence of PLP and reduced levels of MBP in the MD rats point to similarities with the jimpy mouse lesion. Moreover, the defect influences the expression of other myelin proteins and the oligodendrocyte developmental pathway.

The sequence Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly repeats six times serially in the human midsized neurofilament (NF) protein (NF-M). To establish whether Lys-Ser-Pro-Val(Ala) is the major site for in vivo NF phosphorylation, peptides based on the human NF-M repeat were synthesized and chemically phosphorylated. These synthetic peptides were probed with 515 monoclonal antibodies (mAbs) that were raised to, and distinguished, several differentially phosphorylated forms of NF proteins. Studies with 95 of those mAbs that recognized the peptides before and after chemical phosphorylation demonstrated that a highly immunogenic epitope shared by the peptides is present in NFs from all species tested, including invertebrates. This suggests the phylogenetic conservation of a major NF phosphorylation site. Lastly, a cross-reactive antigenic determinant shared by the peptides and the major NF phosphorylation site was shown to exist in neurofibrillary tangles of patients with Alzheimer disease as well as in two neuron-specific microtubule-associated proteins (MAPs)--i.e., MAP2 and tau.

The control of neurofilament (NF) protein gene expression was studied by determining and comparing the levels of mRNA to the heavy (NF-H), mid-sized (NF-M) and light (NF-L) NF protein subunits in rat dorsal root ganglia (DRG) following sciatic nerve transection. mRNA to NF-H (4.5 kb), to NF-M (3.4 kb) and to NF-L (2.5 and 4.0 kb) were identified in Northern blots and quantitated in dot blot analyses, using specific cDNA probes for each NF protein. Following transection, there was a coordinated decline in mRNA levels to all 3 NF proteins, beginning as early as 3 days following transection and continuing for at least 28 days. The early and co-terminal fall in mRNAs suggests that the 3 NF genes are regulated by common factor(s) and that the function of these factor(s) is influenced by the state of axonal continuity with the target organ.

The developmental expression of the myelin basic protein (MBP) gene was studied in rat cultured oligodendrocytes using immunofluorescence and in situ hybridization. In newborn rat brain cultures, which contain only glial cells, large amounts of MBP-specific mRNA (as assayed by grain counts in autoradiograms) abruptly accumulated within immature oligodendrocytes 5 to 6 days postnatal. MBP always emerged 6 to 8 days after birth; thus, a week after, galactocerebroside (GC), an early oligodendrocyte marker, had appeared. The percentage of MBP mRNA and MBP-positive cells peaked at about 15 days postnatal and decreased thereafter. The time of emergence of MBP in these cultured oligodendrocytes appears to be determined at a very early stage in their development and independent of continuous neuronal influences. There is a striking correspondence between the times of appearance of MBP in cultured oligodendrocytes and those in the intact animal. Thus, primary cultures made from 5-day prenatal, newborn, and 2-day postnatal animals all express MBP at about the same developmental stage, namely, after 14, 8, and 6 days in culture, respectively. Furthermore, cultured oligodendrocytes obtained from the spinal cord express MBP before those obtained from midbrain or hemispheres, as they would in the intact animal. Thus, the developmental expression of the MBP gene occurs in a similar time frame in vitro and in vivo. In oligodendrocyte-enriched cultures, where 60% to 80% of the cells express MBP, in situ hybridization with the cDNA clone revealed MBP-specific mRNA in the cell body and sometimes in the processes of the differentiated oligodendrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

The genome of a defective interfering particle (DILT) derived from the heat-resistant strain of vesicular stomatitis virus is expressed in vivo without the assistance of infectious helper virus. The rates of RNA synthesis in the presence of cycloheximide (primary transcription) are the same when infections are with equal numbers of physical particles of DILT or virus. With this treatment, DILT synthesizes only 12-17S mRNAs as characterized by size, polarity, and polyadenylylation. In the absence of cycloheximide, DILT-infected cells produce not only these mRNAs but also a 28S RNA species. This RNA, which represents one half of the viral specific RNA, contains newly synthesized full-length (+) and (-) strand DILT RNA. Both strands are found intracellularly as ribonucleoprotein complexes. Without cycloheximide present, the rate of RNA synthesis by DILT was less than that by virus. This curtailment is most likely due to the inability of DILT to synthesize L protein mRNA. An expanded role for defective interfering particles in infection is discussed.

Dissociated neuron cultures of mice were inoculated with vesicular stomatitis virus (VSV) and subsequently fed with medium containing sufficient antiviral antibody (AB) to neutralize all free virus. In contrast to control acute neuronal infection, which lasts one to two days, AB-treated neuron cultures were maintained as long as two weeks. This protection was not obtained in infected monkey kidney cells treated with AB. Protected neuron cultures were examined with immunolabeling and EM techniques. During the first days of AB treatment, viral buds and surface antigens were often grouped in clusters instead of being diffuse on the neuronal surface. In addition, phagocytic cells often in contact with viruses released by neurons progressively engulfed aggregates of these viruses in their lysosomal systems. Dendritic processes in AB-treated cultures contained more viral antigen and ribonucleoproteins, but less assembly sites than in acutely-infected neurons. However, budding sites were frequent on the side of the post-synaptic density and some viruses seemed to enter directly into the lateral side of the presynaptic terminal to which the productive dendrite was connected. Removal of AB at this stage resulted in reactivation of viral infection in more neurons than those originally infected, but complete viral maturation and release occurred only 2 to 3 days after removal of AB. Chance of reactivation decreased with increasing length of AB treatment, and no viral antigens or budding sites were detected during the second week of AB treatment. Continuous treatment of VSV-infected neuron cultures with antiviral AB first induces (1) activation of non-neuronal cells to phagocytize clusters of viruses forming at the neuron surface, and (2) restriction of virus maturation sites mostly to the postsynaptic area where virus spreading to presynaptic endings seems favored. Prolonged treatment with AB later results in the apparent curing of the infection.

The purpose of this paper is to describe the immunocytochemical-localization of N and NS nucleocapsid proteins of vesicular stomatitis virus in the cells throughout the infectious cycle. N protein was detected in the cytoplasm at 2 h after infection and formed small cytoplasmic clusters which progressively increased in size and number. At 5-6 h, it formed large cytoplasmic inclusions. NS protein was detected in the cytoplasm a little later than N protein and showed almost the same immunostaining pattern. However, diffuse background staining of NS protein was identified throughout the cytoplasm by double immunostaining methods. At electron microscopic level, N protein was mostly granular and occasionally organized in strands at 2-3 h. At 5-6 h, numerous immunostained reaction products were organized in strands. The reaction products of NS protein were almost the same as those of N protein with the exception that diffuse background staining was observed. Cos cells, transfected with SV40 vector containing N gene obtained by recombinant DNA technique, showed clusters of N protein, but virtually no strand at electron microscopic levels. The rapid-freezing and deep-etching replica method demonstrated that loosely coiled VSV genome coated with N protein was localized on cytoplasmic sides of cell membranes in the infected cells. These results showed that complete virus genome replication was needed for strand formation of N and NS proteins and suggested that they were bound to VSV genomes in the infected cells.

We have prepared polyclonal antibodies to the cytoplasmic portion of the envelope glycoprotein G of vesicular stomatitis virus (VSV) by using synthetic peptides corresponding to either the 22 or 11 ultimate carboxy-terminal residues of the G as immunogens. When antibodies to the 22 residue peptide are microinjected into monolayer baby hamster kidney cells before or shortly after infection with wild-type VSV, G protein accumulates in large intracellular patches and little G is observed in the Golgi complex or at the cell surface. In contrast, when antibodies to the 11 residue peptide are injected, no such patches are observed and G protein is seen colocalized with the injected antibody at the endoplasmic reticulum, in the Golgi complex, in transport vesicles, and at the plasma membrane. Microinjection of these antibodies does not disturb the pathway or kinetics of G-protein transport. In cells infected with a temperature-sensitive mutant of VSV, 045, the glycoprotein accumulates in the endoplasmic reticulum at 39.8 degrees C, but rapidly moves through the Golgi apparatus and then to the cell surface after a temperature shift-down to 32 degrees C. Using rhodamine-coupled antibodies to the 11 residue peptide, a microscope stage equipped for precise temperature control, and a silicon intensifier target video camera, we can visualize by video light microscopy the synchronized exocytotic transport of the G protein directly in the living cell.

Most vesicular stomatitis virus (VSV) particles in situ and in vitro after mild treatment appear bacilliform rather than bullet-shaped. However, the nucleocapsid is bullet-shaped. Budding begins with the hemisphere containing the rounded end of the nucleocapsid and finishes with the completion of the nonnucleocapsid-containing “terminal hemisphere”. The procedures commonly used for concentrating and staining the virus in vitro disrupt the fragile terminal hemisphere of the bacilliform causing a collapse of part of its membrane over the planar end of the nucleocapsid. Although the possibility cannot be excluded that some VSV particles are pinched off from the plasma membrane of the cell in the shape of a bullet, we conclude that most of the bullet-shaped virions seen in vitro are an artifact resulting from the processing of the virus for visualization in the electron microscope.

Development in Drosophila is critically dependent on the expression of classes of regulatory genes which establish the polarity of the organism (rev. Niisslein-Volhard et al.), define the segmentation (rev. Scott and Carroll), determine individual segmental characteristics (rev. Gehring), and establish neuronal identities (Doe et al.). These genes were discovered by the effects of mutations which disturbed embryogenesis. Many of the genes which take part in these processes in Drosophila have been characterized at the molecular level and, surprisingly, were found to share a 180 base pair region which is called the homeobox (Gehring). The homeobox encodes a sequence specific DNA binding region, the homeodomain, of these regulatory proteins which presumably modulate the expression of other genes in a coordinated fashion (Gehring; Scott and Carroll). Tremendous excitement was generated by the discovery that the homeobox is highly conserved not only between many developmentally important Drosophila genes, but also among many other organisms including sea urchins (Dolecki et al.), fish (Eiken et al.), frogs (Muller et al.), mice (McGinnis et al., Colberg-Poley et at. 1985a) and humans (Acampora et al.).

Vesicular stomatitis virus (VSV) and defective interfering (DI) particle RNAs were labeled at their 3' ends by using RNA ligase and cytidine 3',5'-bis[32P]phosphate. The RNAs were subjected to partial digestion with alkali and analyzed by oligonucleotide fingerprinting in two dimensions. VSV and DI particle RNAs have complete sequence homology for the first eight bases from the 3' end. The following four positions contain three mismatched nucleotides in which guanosine residues in one strand are replaced by uridine residues in the other. There is again complete homology for the next five bases (positions 13-17). The locations of purine residues within the sequence were confirmed by partial digestion with RNase T1 and RNase U2 and separation by size on 20% acrylamide gels. The latter method also indicated that sequences of VSV and DI particle RNAs diverge beyond the 18th nucleotide from the 3' termini.

The 3′ terminal sequences of four different DI particle RNAs ranging in size from 10S to 30S have been determined directly using rapid RNA sequencing methods or deduced, in the case of the fourth DI RNA, from the complementary sequence of a small RNA transcribed from this part of the genome (Schubert et al., 1978). One DI particle (DI 011) contains covalently linked genomic and antigenomic RNA. The 5′ end of this RNA is identical to that of VSV RNA, as determined by annealing for at least 1 kb, as well as to the other DI particle RNAs used in this study. The 3′ ends of the other three DI particle RNAs are exact copies of the common 5′ terminal sequence for 48 nucleotides in two cases and 45 nucleotides in the third. Beyond these complementary regions the sequences are different for each DI RNA. The fact that these regions differ in length by only three nucleotides, despite the wide differences in the overall size of the DI particle RNAs, indicates that if these DIs were formed by the copy-back mechanisms similar to those proposed by Leppert, Kort and Kolakofsky (1977) and Huang (1977), a specific recognition site for the RNA polymerase must be involved in copying the 5′ terminus. We determined the 5′ terminal sequence from position 43–48 at the end of the complementary region and found it to be 5′-GGUCUU-3′. This hexamer is also part of other highly conserved terminal RNA polymerase initiation sites (Keene et al., 1978; Keene, Schubert and Lazzarini, 1979) and may be a specific internal RNA polymerase recognition site. We conclude that this sequence is one of the elements involved in the genesis of DI particle chromosomes containing short complementary sequences at their termini. The ability of the polymerase to resume synthesis at or near a specific recognition site is discussed.

The amounts of progeny infective (B) and defective (DI) vesicular stomatitis virus (VSV) particles released from cells undergoing standard and autointerfered infections were measured. In experiments using DI 011 , an unusual DI particle containing self-complementary RNA, the maximum yield of progeny DI particles was obtained from cultures infected with so little DI that they showed no signs of autointerference (measured as the reduced yield of standard-size virus or plaque-forming units). Increasing the amount of infecting DI particles above this level did not appreciably affect the yield of DI particles, but did severely diminish the number of infectious progeny particles. The helper function necessary for the replication of the Indiana serotype DI particles which we have used can be supplied by the New Jersey serotype VSV. Surprisingly, much greater amounts of progeny DI particles are obtained from this heterotypic infection than from the homotypic one. Despite the high level of DI replication, very little interference is observed. These results indicate that DI 011 replication and autointerference are not mutually dependent phenomena.

Infection of dissociated neuron cultures of mice with VSV and its defective particle DI-T was studied using fluorescent light microscopy as well as transmission and scanning electron microscopy. When cultures are infected with wild virus, VSV replicates selectively in neurons, producing cell death within 24-48 hr. Sensory and immature neurons express viral antigen most rapidly. Viral antigen and viral budding sites are detected along the neuron soma and dendrites. When large amounts of DI-T particles are added to the wild virus inoculum, viral growth is completely suppressed in mature neurons, the cell killing effects of VSV are considerably delayed and co-infected cultures survive for 5-16 days. Viral antigen accumulates in cytoplasmic inclusions and on the membrane of neuron cell somas and dendrites in the virtual absence of viral assembly. Identical modulation of VSV infection in mature neuron cultures is obtained when DI-T particles are added before or after the wild virus, but ultraviolet inactivation of DIs completely abolishes their protective effect. Immature neurons or Vero cells cannot be protected from acute cytopathic changes by an equivalent amount of DI particles. Thus DIs interfere with replication and assembly of the wild virus and attenuate cell killing effects in mature neurons in vitro.

The effect of chloramphenicol on the synthesis and accumulation of ribonucleic acid (RNA) in Bacillus subtilis was studied. In the presence of chloramphenicol, transfer RNA and ribosomal RNA were synthesized as rapidly 2 to 3 hr after challenge as they were just prior to the addition of the antibiotic. However, under the same conditions, net RNA accumulation ceased after only 30 to 45 min. The failure to accumulate RNA after this time resulted from a rapid degradation of ribosomal RNA synthesized in the presence of chloramphenicol and a slow degradation of mature ribosomes. Since transfer RNA was not appreciably degraded, the ratio of transfer RNA to total RNA increased during the challenge.

The changes in the syntheses of macromolecules that aecompany sporulation of Bacillus subtilis have been the subject of several recent reports. In brief, the available data show that as a rapidly growing culture of B. sublilis departs from strict logarithmic growth, the net synthesis of RNA is abruptly arrested although the protein and DNA contenit approximately double after that time.' De novo RNA synthesis, however, measured by the incorporationi of labeled precursors, coiitinues throughout the stationary and sporulation phase but is just offset by a degradation of cellular RNA. The IRNA synthesized during this time appears not only in the messeniger fraction but also in soluble and ribosomal fractioiis.'-3 Despite this active turniover of soluble and ribosomal IRNA durilng sporulation, the RNA composition of B. subtilis spores appears to be only slightly different from that of vegetative cells. The ratio of soluble to ribosomal RNA increases during stationary phase, but the base compositions of these species are not signifieantly different from their counterparts in vegetative cells.4I However, Doi6 has observed an asymmetry between the elution profiles from methylated albumin kieselguhr columns of soluble RNA from vegetative cells and spores, indicating an alteration of this fraction during sporulation. In the present communication, the abilities of sRNA from spores and vegetative cells of B. subtilis to accept several amino acids are compared. Of the six amino acids tested, all but lysine were incorporated into spore sRNA to a lesser extent than into vegetative sRNA. The incorporation of lysine into sporal sRNA was more than twice that observed when vegetative sRNA was employed as an aeceptor. Comparison of the elution profiles of spore and vegetative lysyl-sRNA shows that spore sRNA contains a component not found in appreciable amounts in vegetative slRNA.

Development of poly(ethylene)imine cellulose thin layers with phosphate solutions gives improved resolution of complex mixtures of nucleotides. Phosphate development also minimizes the tailing of highly radioactive orthophosphate present in the mixtures and thus facilitates chromatographic analysis of crude acid extracts of phosphate-labeled bacteria. Conditions employing phosphate development are described which give semi-quantitative resolution of the ribonucleoside triphosphate components of such extracts after one-dimensional chromatography as well as two-dimensional systems for quantitative resolution of the major nucleotide components.

The proteolipid protein gene products DM-20 and PLP are adhesive intrinsic membrane proteins that make up ≥50% of the protein in myelin and serve to stabilize compact myelin sheaths at the extracellular surfaces of apposed membrane lamellae. To identify which domains of DM-20 and PLP are positioned topologically in the extracellular space to participate in adhesion, we engineered N-glycosylation consensus sites into the hydrophilic segments and determined the extent of glycosylation. In addition, we assessed the presence of two translocation stop-transfer signals and, finally, mapped the extracellular and cytoplasmic dispositions of four antibody epitopes. We find that the topologies of DM-20 and PLP are identical, with both proteins possessing four transmembrane domains and N and C termini exposed to the cytoplasm. Consistent with this notion, DM-20 and PLP contain within their N- and C-terminal halves independent stop-transfer signals for insertion into the bilayer of the rough endoplasmic reticulum duringde novosynthesis. Surprisingly, the conformation (as opposed to topology) of DM-20 and PLP may differ, which has been inferred from the divergent effects that many missense mutations have on the intracellular trafficking of these two isoforms. The 35 amino acid cytoplasmic peptide in PLP, which distinguishes this protein from DM-20, imparts a sensitivity to mutations in extracellular domains. This peptide may normally function during myelinogenesis to detect conformational changes originating across the bilayer from extracellular PLP interactionsin transand trigger intracellular events such as membrane compaction in the cytoplasmic compartment.