Observations on recovery from and recurrence of HSV-2 infections in adult mice that were rescued from lethal vaginal infection by antiviral therapy.

An adult mouse model for studies of latency and recurrence after vaginal HSV-2 infection is not available at present, largely because the infection kills most mice within 14 days. We describe here an antiviral therapy that rescues most vaginally infected mice from death. Vaginally infected mice were nearly all rescued by combined treatment with one dose of monoclonal anti-HSV glycoprotein D 3 days after infection plus valacyclovir in the drinking water on days 3, 4, 5, 7, 9, 11, 13, and 15 after infection. At 60 days after infection, PCR measurements revealed that most rescued mice had viral DNA in their lumbosacral dorsal root ganglia, lumbosacral spinal cords, and paracervical autonomic ganglia, consistent with the possibility that latent infections were established. At this time, immunolabeling revealed CD45+ lymphoid cells in these neural tissues in rescued mice but not in normal control mice. In vivo depletion of T lymphocytes with monoclonal antibodies caused a recurrence of herpes illness symptoms earlier and in a larger proportion of rescued mice than was observed in non-depleted rescued mice. Interestingly, many rescued mice (46/114) spontaneously developed a syndrome of typical herpes illness symptoms that began with ruffled fur on a mouse that previously had sleek fur and progressed to arched backs, feeble gait, hindlimb paralysis, and death or euthanasia, or in some cases to recovery to health. This high incidence of apparent spontaneous recurrence of HSV-2 infection in rescued mice suggests that it may be possible, with some refinement of the procedure, to obtain an effective adult mouse model for studies of therapeutic vaccination to inhibit or prevent HSV-2 recurrence after genital tract infection.

Cloning and characterization of a functional P2X receptor from larval bullfrog skin.

ATP activates an apical-to-basolateral nonselective cation current across the skin of larval bullfrogs (Rana catesbeiana) with similarities to currents carried by some P2X receptors. A functional P2X receptor was cloned from tadpole skin RNA that encodes a 409-amino acid protein with highest protein homology to cP2X(8). RT-PCR showed that this transcript was found in skin, heart, eye, brain, and skeletal muscle of tadpoles but not in skin, brain, or heart of adults. After transcribed RNA from this clone was injected into Xenopus oocytes, application of ATP activated a transient current similar to other P2X receptors and the ATP-activated transient in short-circuit current (I(sc)) across intact skin. The agonists 2-methylthio-ATP and adenosine-5'-O-(thiotriphoshate) also activated transient currents. alpha,beta-Methylene-ATP and ADP were poor agonists of this receptor. Suramin and pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS) were potent antagonists, and PPADS showed an irreversible blockade of this receptor to agonist activation. Under external Na(+)-free, Ca(2+)/Mg(2+)-free conditions (N-methyl-D-glucamine replacement, 0.5 mM EGTA), ATP activated a steadily increasing inward current. Fluorescence microscopy showed that propidium was entering the cells, suggesting that a relatively large pore size was formed under zero divalent conditions. This clone has some characteristics consistent with previously described ATP-activated I(sc) in the tadpole skin. Because the clone is not found in adult skin, it may have some exclusive role in the tadpole such as sensory reception by the skin or triggering apoptosis at metamorphosis.

The SAND domain structure defines a novel DNA-binding fold in transcriptional regulation.

The SAND domain is a conserved sequence motif found in a number of nuclear proteins, including the Sp100 family and NUDR. These are thought to play important roles in chromatin-dependent transcriptional regulation and are linked to many diseases. We have determined the three-dimensional (3D) structure of the SAND domain from Sp100b. The structure represents a novel alpha/beta fold, in which a conserved KDWK sequence motif is found within an alpha-helical, positively charged surface patch. For NUDR, the SAND domain is shown to be sufficient to mediate DNA binding. Using mutational analyses and chemical shift perturbation experiments, the DNA binding surface is mapped to the alpha-helical region encompassing the KDWK motif. The DNA binding activity of wild type and mutant proteins in vitro correlates with transcriptional regulation activity of full length NUDR in vivo. The evolutionarily conserved SAND domain defines a new DNA binding fold that is involved in chromatin-associated transcriptional regulation.

Nuclear DEAF-1-related (NUDR) protein contains a novel DNA binding domain and represses transcription of the heterogeneous nuclear ribonucleoprotein A2/B1 promoter.

Nuclear DEAF-1-related (NUDR) protein is a novel transcriptional regulator with sequence similarity to developmental and oncogenic proteins. NUDR protein deletions were used to localize the DNA binding domain between amino acids 167 and 368, and site-specific DNA photocross-linking indicated at least two sites of protein-DNA contact within this domain. The DNA binding domain contains a proline-rich region and a region with similarity to a Myc-type helix-loop-helix domain but does not include the zinc finger motif at the C terminus. Deoxyribonuclease I protection assays confirmed the presence of multiple NUDR binding motifs (TTC(C/G)G) in the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) promoter and also in the 5'-untranslated region (UTR) of hNUDR cDNA. NUDR produced a 65-70% repression of the hnRNP A2/B1 promoter activity, and NUDR binding motifs in the 5'-UTR were found to mediate this repression. NUDR-dependent repression was also observed when the 5'-UTR of NUDR was placed onto a heterologous thymidine kinase promoter in an analogous 5'-UTR position but not when placed upstream of transcription initiation. These results suggest that NUDR may regulate the in vivo expression of hnRNP A2/B1 and NUDR genes and imply that inactivation of NUDR could contribute to the overexpression of hnRNP A2/B1 observed in some human cancers.

Characterization of a nuclear deformed epidermal autoregulatory factor-1 (DEAF-1)-related (NUDR) transcriptional regulator protein.

A monkey kidney cDNA that encodes a nuclear regulatory factor was identified by expression and affinity binding to a synthetic retinoic acid response element (RARE) and was used to isolate human placental and rat germ cell cDNAs by hybridization. The cDNAs encode a 59-kDa protein [nuclear DEAF-1-related (NUDR)] which shows sequence similarity to the Drosophila Deformed epidermal autoregulatory factor-1 (DEAF-1), a nonhomeodomain cofactor of embryonic Deformed gene expression. Similarities to other proteins indicate five functional domains in NUDR including an alanine-rich region prevalent in developmental transcription factors, a domain found in the promyelocytic leukemia-associated SP100 proteins, and a zinc finger homology domain associated with the AML1/MTG8 oncoprotein. Although NUDR mRNA displayed a wide tissue distribution in rats, elevated levels of protein were only observed in testicular germ cells, developing fetus, and transformed cell lines. Nuclear localization of NUDR was demonstrated by immunocytochemistry and by a green fluorescent protein-NUDR fusion protein. Site-directed mutagenesis of a nuclear localization signal resulted in cytoplasmic localization of the protein and eliminated NUDR-dependent transcriptional activation. Recombinant NUDR protein showed affinity for the RARE in mobility shifts; however it was efficiently displaced by retinoic acid receptor (RAR)/retinoid X receptor (RXR) complexes. In transient transfections, NUDR produced up to 26-fold inductions of a human proenkephalin promoter-reporter plasmid, with minimal effects on the promoters for prodynorphin or thymidine kinase. Placement of a RARE on the proenkephalin promoter increased NUDR-dependent activation to 41-fold, but this RARE-dependent increase was not transferable to a thymidine kinase promoter. Recombinant NUDR protein showed minimal binding affinity for proenkephalin promoter sequences, but was able to select DNA sequences from a random oligonucleotide library that had similar core-binding motifs (TTCG) as those recognized by DEAF-1. This motif is also present between the half-sites of several endogenous RAREs. The derived consensus- binding motif recognized by NUDR (TTCGGGNNTTTCCGG) was confirmed by mobility shift and deoxyribonuclease I (DNase I) protection assays; however, the consensus sequence was also unable to confer NUDR-dependent transcriptional activation to the thymidine kinase promoter. Our data suggests that NUDR may activate transcription independently of promoter binding, perhaps through protein-protein interaction with basal transcription factors, or by activation of secondary factors. The sequence and functional similarities between NUDR and DEAF-1 suggest that NUDR may also act as a cofactor to regulate the transcription of genes during fetal development or differentiation of testicular cells.

Prodynorphin gene expression in the rat intermediate pituitary lobe: gender differences and postpartum regulation.

The distribution of prodynorphin (proDyn) messenger RNA (mRNA) was examined in the rat pituitary using Northern and in situ hybridization analysis. Anterior pituitary gonadotrophs are known to express ProDyn, but the present study demonstrated that proDyn mRNA was also expressed in the intermediate lobe melanotrophs and was colocalized with POMC mRNA. The 2.6-kilobase proDyn transcript observed in the intermediate lobe was shown to be translatable by polysome analysis. Immunohistochemical studies showed dynorphin (Dyn)-like immunoreactivity in all intermediate lobe melanotrophs. Intermediate lobe proDyn gene expression was not regulated by dopamine, in contrast to intermediate lobe POMC mRNA levels, which were increased with haloperidol and decreased with bromocriptine treatment, as expected. A gender difference in ProDyn gene expression was noted, since intermediate lobes of male rats had nearly 2-fold higher proDyn mRNA levels than intermediate lobes of female rats. In contrast, no gender difference of intermediate lobe POMC mRNA levels were detected. ProDyn mRNA levels were up-regulated by 3- to 4-fold in the intermediate lobes of postpartum females as compared to pregnant or nonpregnant female rats, whereas POMC mRNA levels were unchanged, suggesting a role for intermediate lobe ProDyn in the postpartum period of the female rat. Although our results demonstrate proDyn and POMC coexpression in the pituitary intermediate lobe melanotrophs and show a differential regulational control for each gene in this tissue, the present data also strengthen the notion that proDyn is a precursor that has a role to play in reproductive functions.

Germ cell localization of a testicular growth hormone-releasing hormone-like factor.

GH-releasing hormone (GHRH)-like mRNA and immunoreactivity (t-GHRH-li) are present in the testes of rats and humans. To learn more about the physiology of t-GHRH-li mRNA, we performed a series of experiments that disrupted various testicular functions. We employed ethylene dimethanesulfonate, a Leydig cell toxin, to assess the effects of Leydig cell ablation on t-GHRH-li mRNA and protein levels in prepubertal and postpubertal male rats. The ethylene dimethanesulfonate treatment resulted in decreases in serum testosterone, but had no effect on t-GHRH-li mRNA or peptide levels. To assess the effect of GHRH on Leydig cell steroidogenesis, Leydig cells were isolated by Percoll gradient centrifugation and cultured in the presence or absence of hCG, GHRH, or hCG plus GHRH. GHRH had no effect on steroidogenesis by Leydig cells, either alone or in combination with hCG. To localize t-GHRH-li mRNA within rat testis, in situ hybridization analysis was performed on testicular sections from normal rats, using a [35S]GHRH riboprobe. Grains were detected in spermatogenic cells with the antisense probe, whereas none was detected with the sense strand (control) probe. To verify these results, Northern blot analysis of RNA from separated testicular cells was performed. t-GHRH-li mRNA was detected in spermatocytes and round spermatids and to a lesser extent in Sertoli cells, but not in elongating spermatids, Leydig cells, or peritubular myoid cells. t-GHRH-li mRNA was also not found in epididymis. Since our experiments localized t-GHRH-li mRNA to spermatogenic cells, methoxyacetic acid (MAA), a pachytene spermatocyte toxin, was administered to postpubertal rats to determine whether t-GHRH-li is expressed primarily in pachytene spermatocytes. MAA treatment caused a decrease in testicular weight, which gradually returned to control levels by 42 days. Serum FSH levels in the treated animals fluctuated over the course of the experiment, while those in control animals remained steady. However, there was no difference in testicular GHRH-li mRNA levels between control and treated animals at any treatment time. Insulin-like growth factor-I and -II mRNA levels were also unaltered by the MAA treatment. We conclude from these results that t-GHRH-li is synthesized in early spermatogenic cells, but not in mature sperm, and that testicular GHRH-li does not play a major role in steroidogenesis by the Leydig cell.

Modification of the retinoic acid signaling pathway by the catalytic subunit of protein kinase-A.

Retinoic acid receptors (RARs) are ligand-activated nuclear transcription factors that belong to the steroid-thyroid hormone receptor superfamily. We have used the transient transfection of a retinoic acid-responsive reporter plasmid (RARECAT) to investigate the potential interactions between the retinoic acid (RA) and cAMP signaling pathways. Cotransfections of expression plasmids for the catalytic (C) subunits of cAMP-dependent protein kinase with RARECAT showed ligand-independent activation in both CV-1 and HeLa cells and a further 2-fold increase in RARECAT activity in the presence of RA. In CV-1 cells, cotransfections of the expression plasmids for RAR and the C-subunits produced increases in RARECAT activity (12- and 8-fold in the absence of ligand and 21- and 15-fold in the presence of RA for the C alpha- and C beta-isoforms, respectively). Cotransfections of both the C beta-subunit and RAR expression plasmids in HeLa cells produced 22- and 114-fold increases in RARECAT activity in the absence and presence of RA, respectively. These results provide evidence to suggest that the RA and cAMP signaling pathways are coupled, and signaling cross-talk may occur through the direct phosphorylation of RARs by the C-subunit as indicated by in vitro phosphorylation of the receptor.

Testicular expression of PC4 in the rat: molecular diversity of a novel germ cell-specific Kex2/subtilisin-like proprotein convertase.

The rat cDNA sequence of PC4 (rPC4), representing a new member of the Kex2/subtilisin-like proprotein convertases, demonstrated the presence of at least three rPC4 mRNAs resulting in the production of rPC4-A (654 amino acids), rPC4-B (619 amino acids), and rPC4-C (609 amino acids) with different C-terminal sequences. Analogous to rat PC4, three cDNAs were also found for the mouse PC4. The observed molecular diversity of PC4 mRNA possibly results from the differential splicing and/or exon skipping of the parent gene. PC4 mRNA, with a major form at 2.8 kilobases, was highly abundant in the rat testis but could not be detected by Northern analysis in any other tissues including the central nervous system and peripheral tissues. Testicular cell separation studies combined with Northern analysis indicate the high expression levels of PC4 in germ cells but not in Leydig, Sertoli, or peritubular cells. In situ hybridization histochemistry confirms the site of PC4 gene expression as the pachytene spermatocytes and the round spermatids but not in the elongating spermatids. We also demonstrate the colocalization of PC4 with proenkephalin in testicular germ cells by in situ hybridization. A study of the ontogeny of PC4 indicated that PC4 mRNA was first expressed postnatally between days 19 and 22, coinciding with the first stages of spermiogenesis. The stage-specific expression of PC4 in testis indicates its potential role in the developmental maturation of germ cells and that this convertase may play a specific physiological function in reproduction.

Glutamate decarboxylases in nonneural cells of rat testis and oviduct: differential expression of GAD65 and GAD67.

gamma-Aminobutyric acid (GABA) and its synthetic enzyme, glutamate decarboxylase (GAD), are not limited to the nervous system but are also found in nonneural tissues. The mammalian brain contains at least two forms of GAD (GAD67 and GAD65), which differ from each other in size, sequence, immunoreactivity, and their interaction with the cofactor pyridoxal 5'-phosphate (PLP). We used cDNAs and antibodies specific to GAD65 and GAD67 to study the molecular identity of GADs in peripheral tissues. We detected GAD and GAD mRNAs in rat oviduct and testis. In oviduct, the size of GAD, its response to PLP, its immunoreactivity, and its hybridization to specific RNA and DNA probes all indicate the specific expression of the GAD65 gene. In contrast, rat testis expresses the GAD67 gene. The GAD in these two reproductive tissues is not in neurons but in nonneural cells. The localization of brain GAD and GAD mRNAs in the mucosal epithelial cells of the oviduct and in spermatocytes and spermatids of the testis shows that GAD is not limited to neurons and that GABA may have functions other than neurotransmission.

Regulation of the human enkephalin promoter by two isoforms of the catalytic subunit of cyclic adenosine 3',5'-monophosphate-dependent protein kinase.

Cyclic AMP regulates a variety of cellular responses through activation of the catalytic subunit of cAMP-dependent protein kinase. The cDNAs for two protein isoforms of the catalytic subunit, C alpha and C beta, were placed into expression vectors, and their ability to stimulate cAMP-dependent transcription of the human enkephalin promoter was examined in transiently transfected CV-1 cells. Expression vectors for C alpha and C beta that were directed by the human cytomegalovirus promoter produced up to 350- and 200-fold increases in chloramphenicol acetyltransferase activity, respectively, when cotransfected with the ENKAT-12 reporter plasmid. Transcriptional activation was shown to be dependent upon functional kinase activity by point mutations in catalytic subunit vectors which eliminated activation. Transcriptional activation by C alpha and C beta was eliminated when the cAMP response elements (CREs) were deleted from the native enkephalin promoter, but activation was recovered when this region was replaced with an oligonucleotide containing two copies of the somatostatin CRE consensus TGACGTCA. C alpha expression vectors were found to produce 2-fold greater transcriptional activation than C beta expression vectors. These results were most likely due to the cellular kinase activity produced by the catalytic subunit expression vectors and did not appear to be dependent on CRE motif or substrate specificity. In vitro mutagenesis indicates that neither C alpha nor C beta requires N-terminal myristylation for transcriptional activation, but threonine-197 is critical to subunit function.

A transferrinlike (hemiferrin) mRNA is expressed in the germ cells of rat testis.

In the testis, germ cells which are separated from the serum by the blood-testis barrier rely primarily on the Sertoli cell to obtain nutrients. For example, transferrin synthesized by the Sertoli cell is important in delivering iron from the serum to the developing germ cells. Because of its role in the testis, Sertoli cell transferrin protein and mRNA have been extensively studied. By using RNA blot analysis of rat testicular tissue, we detected a transcript of 2.6 kb which is attributed to transferrin. In addition, we detected a novel mRNA of 0.9 kb which had sequence similarity to the 3' end of transferrin. This 0.9-kb mRNA was present in germ cells, but not Sertoli cells, liver, or brain. The primary source of this mRNA in the testis was round spermatids. Sequence analysis of a cDNA clone showed that this mRNA encoded a protein with sequence similarity to the carboxy terminus of transferrin. Polysome analysis indicated that this transcript was translated and may therefore have importance in the iron metabolism of germ cells. The evolutionary implications between the transferrinlike mRNA germ cells and the gene duplication event which resulted in the diferric binding of transferrin are discussed.

Atypical prodynorphin gene expression in corticosteroid-producing cells of the rat adrenal gland.

Prodynorphin (proDyn) gene expression was examined in the rat adrenal gland. In situ hybridization revealed a heterogenous proDyn mRNA distribution limited almost exclusively to the adrenal cortex; the inner cortical layers contained the highest amounts. In the adrenal medulla, only scattered single cells were seen. By Northern (RNA) blot analysis, adrenocortical proDyn mRNA levels were highly abundant but of smaller size than proDyn transcripts found in the brain. Low levels of the brain-size proDyn mRNA transcript were detected but restricted to the medulla. A discrepancy was suggested when comparing the high abundance of proDyn mRNA levels with the low levels of proDyn-derived peptide in the adrenal. A hypothesis of nontranslation of the shorter proDyn mRNA by adrenocortical cells was rejected because polysomal loading analysis suggests that the mRNA is translated. We propose that adrenocortical proDyn-derived peptides are not targeted for storage but are released shortly after synthesis, thus accounting for low peptide levels. We also measured proDyn mRNA levels in response to stimuli known to affect adrenocortical cells and their most important function--steroidogenesis. Adrenals from hypophysectomized rats had less proDyn mRNA by a factor of 5 than adrenals from normal sham-operated rats. Normal levels were restored by adrenocorticotrophic hormone administration, indicating a potential importance of adrenal proDyn in the hypothalamic-adrenal-pituitary axis.

Sertoli cells are the primary site of prodynorphin gene expression in rat testis: regulation of mRNA and secreted peptide levels by cyclic adenosine 3' ,5'-monophosphate analogs in cultured cells.

Prodynorphin is one of three endogenous opioid peptide genes expressed in testis. Through the use of cell fractionation procedures and Northern blot analysis, Sertoli cells were found to be the primary site of prodynorphin mRNA synthesis in rat testis. In situ hybridization of a prodynorphin cRNA probe to fixed adult tissue confirmed this result. Treatment of primary cultures of rat Sertoli cells with a cAMP analog, 8-(4-chlorophenylthio)cAMP, resulted in a transient 5.6-fold increase in steady state prodynorphin mRNA levels relative to those in control cells. This increase was maximal at 48 h of treatment, after which mRNA levels gradually declined. Treatment of Sertoli cells with cAMP analogs resulted in concurrent 2.6-fold decreases in sulfated glycoprotein-2 mRNA levels. Culture medium from Sertoli cells showed a 3.1-fold increase in secreted dynorphin immunoreactivity after treatment with 8-(4-chlorophenylthio)cAMP. Chromatographic analysis indicates that the majority of the immunoreactive dynorphin peptide synthesized in Sertoli cells is present as high mol wt species, with some processing to bioactive peptides.

Translational control of germ cell-expressed mRNA imposed by alternative splicing: opioid peptide gene expression in rat testis.

The three genes encoding the opioid peptide precursors (prodynorphin, proenkephalin, and proopiomelanocortin) are expressed in the rat testis. The sizes of the three opioid mRNAs in the testis differ from the sizes of the corresponding mRNAs in other rat tissues in which these genes are expressed. The smaller testicular proopiomelanocortin mRNA has previously been demonstrated to arise from alternative transcriptional initiation. In the present study, we found that the smaller testicular prodynorphin mRNA, expressed in Sertoli cells, results from alternative mRNA processing. Exon 2, which makes up 5' untranslated sequence, is removed from the mature transcript. Polysome analysis of brain and testis RNA indicates that the alteration of the prodynorphin leader sequence in the testis-specific transcript does not affect the efficiency of translation of this mRNA. The larger testicular proenkephalin transcript, expressed in developing germ cells, also results from alternative mRNA processing. Alternative acceptor site usage in the splicing of intron A results in a germ cell-specific proenkephalin transcript with a 491-nucleotide 5' untranslated leader sequence preceding the preproenkephalin-coding sequence. Polysome analysis indicates that this germ cell-specific proenkephalin mRNA is not efficiently translated. Mechanisms by which alternative mRNA splicing may serve to confer translational regulation upon the testicular proenkephalin transcript are discussed.

Developmentally regulated gene from Leishmania encodes a putative membrane transport protein.

We have cloned a developmentally regulated gene from the parasitic protozoan Leishmania enrietti. The mRNA from this gene accumulates to a much higher level in the promastigote stage of the parasite life cycle that lives in the gut of the insect vector than in the amastigote stage of the parasite that lives inside the macrophages of the mammalian host. The predicted protein encoded by this gene is homologous to the human erythrocyte glucose transporter and to several sugar-transport proteins from Escherichia coli. These structural similarities strongly suggest that the cloned gene encodes a membrane transport protein that is developmentally induced when the parasite enters its insect vector. Regulated membrane transporters may be required for the parasite to adapt to the environment of the insect gut.

Biosynthesis and molecular cloning of sulfated glycoprotein 1 secreted by rat Sertoli cells: sequence similarity with the 70-kilodalton precursor to sulfatide/GM1 activator.

Sulfated glycoprotein 1 (SGP-1) is one of the abundant proteins secreted by rat Sertoli cells. Pulse-chase labeling shows that SGP-1 is synthesized as a cotranslationally glycosylated 67-kilodalton (kDa) precursor which is posttranslationally modified to a 70-kDa form before secretion to the extracellular space. A plasmid cDNA library was constructed from immunopurified mRNA, and two overlapping clones coding for the entire protein coding sequence were isolated. The cDNAs represent 27 nucleotides of 5' noncoding sequence, 1554 nucleotides of coding sequence, and 594 nucleotides of 3' noncoding sequence. The derived SGP-1 sequence contains 554 amino acids and has a molecular weight of 61,123. Four potential N-glycosylation sites occur within the sequence. An internal region of SGP-1 shows 78% sequence identity with the 67 N-terminal amino acids described for human sulfatide/GM1 activator (SAP-1). Sequence comparisons suggest that SGP-1 is the precursor to sulfatide/GM1 activator; however, the secretion of the protein from Sertoli cells is distinct from the proteolytic processing and lysosomal compartmentalization which have been described for human fibroblasts. The presence of internal sequence similarity suggests that three additional binding sites may occur in SGP-1. Northern blots show similar levels of expression for the 2.6-kilobase SGP-1 mRNA in all tissues examined. The site of SGP-1 synthesis in testis was localized to Sertoli cells by immunofluorescence and in situ hybridization.

Biosynthesis and molecular cloning of sulfated glycoprotein 2 secreted by rat Sertoli cells.

Sulfated glycoprotein 2 (SGP-2) is the major protein secreted by rat Sertoli cells. Pulse-chase labeling shows that SGP-2 is synthesized as a cotranslationally glycosylated 64-kDa precursor that is modified to a negatively charged 73-kDa form before intracellular cleavage to the mature 47- and 34-kDa subunits. A plasmid cDNA library was constructed from immunopurified mRNA, and a recombinant clone containing the entire protein coding sequence of SGP-2 was isolated. The 1857-nucleotide cDNA consists of a 297-nucleotide 5' noncoding segment, a 1341-nucleotide coding segment, and a 219-nucleotide 3' noncoding sequence. The 5' noncoding region contains five ATG codons followed by four short open reading frames. The derived SGP-2 sequence has a molecular weight of 51,379 and contains six potential N-glycosylation sites. Proteolytic processing sites for the preproprotein were determined by amino-terminal sequencing of the isolated SGP-2 subunits. Northern blots show a wide tissue distribution for the 2.0-kb SGP-2 message, and computer sequence analysis indicates a significant relationship between SGP-2 and human apolipoprotein A-I.

Nonaqueous fractionation of HeLa cells in glycols.

A further modification of Behrens ' method of nonaqueous cell fractionation, using glycols as media for homogenization and centrifugation, was presented. HeLa cells were frozen in melting Freon-12 ( CCl2F2 ), dried under vacuum at -30 degrees C, sonicated in hexylene glycol at -35 degrees C, and centrifuged through either propylene glycol or a mixture of the two glycols at -40 degrees C. The centrifugation yielded a nuclear pellet and a cytoplasmic supernatant. The supernatant was recentrifuged at -10 degrees C, yielding a cytoplasmic pellet. The success of the method depended on the temperature-dependent viscosities of the glycols and on the aggregation properties of cell structures in cold glycols. The allowed ranges of low temperatures were critical but not difficult to use; methods are given for sonication and for centrifugation. The two pellet fractions together contained 90% or more of cellular proteins and nucleic acids. Distribution of [3H]uridine-labeled nucleic acids showed that the first pellet (nuclei) contained over 95% of the nuclear markers, DNA, and ribosomal RNA precursors, plus about 10% of the cytoplasmic marker, 18 S ribosomal RNA. The cytoplasmic pellet contained less than 5% of the nuclear markers. Two enzyme activities were tested; DNA polymerase, a mostly soluble nuclear marker frequently eluted in aqueous fractionation, and lactate dehydrogenase, a cytoplasmic marker. The two enzymes each lost activity in propylene glycol but not in a mixture of 90% hexylene glycol and 10% propylene glycol, so the glycol mixture was used as a centrifugation medium when studying enzymes. The glycol mixture sometimes gave more cytoplasmic material, up to 20% of the 18 S ribosomal RNA, in the nuclear pellet. The fractionation showed, as expected, that DNA polymerase activity was 95% nuclear and lactate dehydrogenase activity was more than 68% cytoplasmic. The concentration of cytoplasmic material afforded by the glycol method allowed the detection of a small amount (approx. 5%) of DNA polymerase activity not associated with nuclei. The chief reason for use of the glycol method instead of other methods of cell fractionation is that easily solubilized cellular material can be recovered in concentrated pellet form in the appropriate nuclear or cytoplasmic fraction.