γ-Tubulin is a universal component of microtubule organizing centers where it is believed to play an important role in the nucleation of microtubule polymerization. γ-Tubulin also exists as part of a cytoplasmic complex whose size and complexity varies in different organisms. To investigate the composition of the cytoplasmic γ-tubulin complex in mammalian cells, cell lines stably expressing epitope-tagged versions of human γ-tubulin were made. The epitope-tagged γ-tubulins expressed in these cells localize to the centrosome and are incorporated into the cytoplasmic γ-tubulin complex. Immunoprecipitation of this complex identifies at least seven proteins, with calculated molecular weights of 48, 71, 76, 100, 101, 128, and 211 kD. We have identified the 100- and 101-kD components of the γ-tubulin complex as homologues of the yeast spindle pole body proteins Spc97p and Spc98p, and named the corresponding human proteins hGCP2 and hGCP3. Sequence analysis revealed that these proteins are not only related to their respective homologues, but are also related to each other. GCP2 and GCP3 colocalize with γ-tubulin at the centrosome, cosediment with γ-tubulin in sucrose gradients, and coimmunoprecipitate with γ-tubulin, indicating that they are part of the γ-tubulin complex. The conservation of a complex involving γ-tubulin, GCP2, and GCP3 from yeast to mammals suggests that structurally diverse microtubule organizing centers such as the yeast spindle pole body and the animal centrosome share a common molecular mechanism for microtubule nucleation.

Enzymes that catalyze the two successive stages of Golgi-associated processing of asparagine-linked oligosaccharides distributed differently when membranes from Chinese hamster ovary cells were centrifuged in a sucrose density gradient. A mannosidase that removes only outer, a-1,2-linked mannose residues from the precursor oligosaccharides of the vesicular stomatitis viral G protein (to yield a "trimmed" oligosaccharide core) was separated from enzymes (galactosyl-and sialyltransferases) that act in the later, terminal stage ofglycosylation. Freshly acylated G protein with newly trimmed oligosaccharides banded in the distribution of early-acting membranes, defined by the mannosidase, whereas G protein pulse-labeled with [3H]galactose distributed in the profile of the late-acting membranes. G protein present in the early-acting membranes in crude fractions could be terminally glycosylated by incubation with exogenous Golgi membranes in vitro; G protein lost its ability to be processed in vitro as it appeared to enter the late-acting membranes in vivo. These experiments reveal the existence of two distinct compartments through which intracellularly transported proteins such as G pass in sequence as Golgi-associated processes are carried out. It is likely that these compartments consist of cisternae on the cis and trans sides of the Golgi stack. The stack of cisternae composing the Golgi apparatus is markedly asymmetric (1-7), its cis [or entry (8, 9)] and trans [or exit (9, 10)] faces differing in morphological and histochemical properties. This cis-trans polarity could signify a fundamental division of the stack into functionally distinct compartments between which proteins can be transported vectorially. Alternatively, this overall asymmetry could reflect a continuous gradient ofcomposition, the result ofa sorting process operating within a stack composed of multiple copies of the same kind of compartment.We recently reported (11) a dramatic change in the behavior in a cell-free assay system (12, 13) of a plasma membrane-type glycoprotein [the G protein encoded by vesicular stomatitis virus (VSV)] that occurred as a result of passage through the Golgi. Specifically, the assay distinguished two intracellular pools through which G protein passed in rapid succession: an earlier "transferable" pool that can be donated by membranes from infected cells to exogenous Golgi for oligosaccharide processing in vitro and a later "nontransferable" pool that cannot. The transferable pool appeared to reside in Golgi membranes (11). If the other pool also resided in Golgi membranes, but in a different region, then the change in properties of G protein (transferable-versus nontransferable) in going from one region ofthe Golgi to the other would signify a compartment boundary in between.Asparagine-linked oligosaccharides (such as those ofthe VSV G protein) are processed in two major stages (14-16). First, the precursor oligosaccharide is "trimmed" by the removal of four a-1,2-linked mannose units. Later, terminal glycosyl...

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Neutralizing antibody titers to herpes simplex virus type 1 (HSV-1) and HSV-2 were measured at birth in normal infants and uninfected infants of mothers with genital HSV infections during pregnancy and at the onset of infection in 5 infants with mild infections and 11 infants with severe infections. Thirty-eight percent of premature and 29% of term infants had neutralization titers of <1:5. High titers (,1:40) were found in 55% of infants of mothers with primary infections during pregnancy and in 76% of infants of mothers with recurrent infections. The mean titers to HSV-1 and -2 in 5 infected infants with mild infections were 1:56 and 1:65 at the time of onset of infection, whereas the mean titers in 11 infants with severe infections were 1:11 and 1:12. Six natally exposed infants who remained asymptomatic were also studied and had a mean titer to HSV-1 of 1:85 and to HSV-2 of 1:69. Therefore, infants with high titers of transplacentally derived antibody had a more favorable outcome than infants with lower titers. Ninety-five percent of the infants of mothers with recurrent infections had a Rawls index of more than 85, suggesting that the antibody response was to HSV-2. However, low levels of antibody with this type specificity failed to protect four infants from infection with HSV-2. Augmentation of the neutralization titer to HSV-2 by the amount of complement present in cord serum was less than twofold. The study suggests that the quantity of antibody derived transplacentally affects the outcome of infection after natal exposure to herpes simplex virus. Complete neutralization of virus by antibody may occur in some infants, and prolongation of the incubation period and modification of the infection may occur in others.Genital infections due to herpes simplex virus (HSV) are among the most common venereal diseases identified in patients from middle and upper socioeconomic groups (3, 7). The incidence of natally acquired HSV infections appears to be increasing (L. Corey, personal communication). In about half of the cases in which the baby becomes infected, maternal infection is not suspected (4,14). Data accumulated by Nahmias et al. suggest that the risk of infection in infants exposed to HSV during vaginal delivery is less if the maternal infection is recurrent (4%) than if it is primary (50%) (13). The difference in prognosis for infants of mothers with recurrent infections as compared with those with primary infections may be related to the location and quantity of virus present and to the quantity of maternally derived antibody acquired by the infant. Adams et al. found that virus was present on the cervix in 87% of women with primary infections but in only 4% of those with recurrent infections (1). The presence of virus high in the birth canal in contrast to the exterior of the labia majora, the usual site for recurrent lesions, might also influence morbidity, since inoculation of the baby from a labial lesion might occur less often than from a cervical lesion. HSV is shed in greater quantities and for l...

The transfer of the vesicular stomatitis virus-encoded glycoprotein (G protein) between Golgi populations in fused cells (Rothman, J . E., L. J . Urbani, and R . Brands . 1984 .