Rat Sertoli and spermatogenic cells express a similar gene, and its product is antigenically related to an outer dense fiber-associated protein.

We have previously reported that a heterodimeric protein secreted by rat Sertoli cells is antigenically related to a protein associated with outer dense fibers of the sperm tail. Therefore, we have explored the possibility that Sertoli and spermatogenic cells express a similar gene encoding a homologous protein. A Sertoli cell heterodimeric protein cDNA probe recognizes specific mRNA in pachytene and round spermatids fractionated by centrifugal elutriation; however, this specific mRNA was less prominent than in cultured Sertoli cells. In agreement with these observations, in situ hybridization experiments show that Sertoli cells are predominantly engaged in active heterodimeric protein mRNA synthesis, while meiotic prophase spermatocytes and spermatids also show significant but less abundant specific mRNA. Immunoblotting experiments demonstrate that, while Sertoli cells synthesize a heterodimeric protein consisting of two disulfide-linked components with molecular masses of 45 and 35 kD, both primary spermatocytes and round spermatids synthesize single 30 kD monomers not associated by disulfide linkage but recognized by antisera to Sertoli cell heterodimeric protein. Immunoblotting and immunogold electron microscopic studies show that antisera to Sertoli cell heterodimeric protein recognize a protein associated with outer dense fibers. This immunoreactivity was abolished by a 5-min pronase treatment, without affecting the integrity of outer dense fibers. Results of this study and previous studies demonstrate that both Sertoli and spermatogenic cells express a similar gene and that an antigenically related product encoded by this gene becomes associated with outer dense fibers during their assembly at spermiogenesis.     

The Drosophila tissue polarity gene starry night encodes a member of the protocadherin family.

The tissue polarity genes control the polarity of hairs, bristles and ommatidia in the adult epidermis of Drosophila. We report here the identification of a new tissue polarity gene named starry night (stan). Mutations in this essential gene alter the polarity of cuticular structures in all regions of the adult body. The detailed polarity phenotype of stan on the wing suggested that it is most likely a component of the frizzled (fz) pathway. Consistent with this hypothesis, stan appears to be downstream of and required for fz function. We molecularly cloned stan and found that it encodes a huge protocadherin containing nine cadherin motifs, four EGF-like motifs, two laminin G motifs, and seven transmembrane domains. This suggests that Stan functions in signal reception, perhaps together with Fz.     

Molecular cloning and differential expression of somatic and testis-specific H2B histone genes during rat spermatogenesis

We have cloned cDNA of a testis-specific histone, TH2B (a variant of H2B), and rat somatic H2B gene to investigate regulation of testis-specific histone genes during rat spermatogenesis. The amino acid sequences deduced from DNA sequences show extensive sequence divergence in the N-terminal third of the two histones. The rest is highly conserved. One cysteine residue was found in TH2B. No cysteine is present in somatic histones except in H3 histone. We investigated the expression of TH2B and H2B genes using the regions of sequence divergence as hybridization probes. The TH2B gene is expressed only in the testis, and the expression of this gene is detected 14 days after birth, reaching a maximum at Day 20. The level of H2B mRNA shows a reciprocal pattern. This contrasting pattern can be explained by the gradually changing proportion of spermatogonia and spermatocytes with testicular maturation. In situ cytohybridization studies show that H2B gene is expressed primarily in proliferating spermatogonia and preleptotene spermatocytes, whereas TH2B gene is expressed exclusively in pachytene spermatocytes which first appear in testis about 14 days after birth. H2B and TH2B genes appear to be ideal markers for the study of proliferation and differentiation events in spermatogenesis and their regulatory mechanisms.     

Intracellular protein phosphorylation in oat (Avena sativa L.) protoplasts by phytochrome action. 1. Measurement of action spectra for the protein phosphorylation

The effects of red light and wavelength dependency of the protein phosphorylation in oat protoplasts were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Red light (660 nm) irradiation of the protoplasts increased the phosphorylation of 15 different proteins, and the phosphorylation of 2 proteins (27 KDa, 32 KDa) out of 15 were observed to be dependent on the wavelength of the irradiating light. The phosphorylation densities of these two proteins increased up to two or three hundred percent during a three-minute period of irradiation. The phosphorylation of these two proteins revealed a red/far-red photoreversibility of phytochrome. When a calcium ion chelator (2 mM EGTA) was added into the cell suspension, the phosphorylations of all the proteins were reduced about 200%. These findings suggest that phytochrome action and Ca2+ influx are certainly involved in the in vivo phosphorylation of proteins in oat protoplasts.     

Repression of Asolectin-Dependent Activation of Partially Lipid Depleted ATPase Prepared from the Plasma Membrane-Enriched Fraction of Cucumber Roots due to Ca2+ Starvation

The ATPase activity of the plasma membrane-enriched fractionwas severely inhibited by withdrawal of Ca²⁺ from the mediumfor 5 days, although the root system appeared to be unaffectedto visual inspection. Partially lipid-depleted ATPases withsimilar ratios of phospholipid to protein were prepared fromthe plasma membrane-enriched fraction of cucumber roots culturedwith control medium and one lacking Ca²⁺, and their propertieswere compared. SDS disc polyacrylamide gel electrophoresis showedthat the polypeptide components were essentially similar betweencontrol and Ca²⁺-starved roots. Partially lipid-depleted ATPasereassociated with asolectin, the lecithin from soybean, showedtypical characteristics of plasma membrane type ATPase; pH optimumat 6.5, high specificity for ATP as substrate and strong inhibitionby vanadate but not nitrate. The activity of reassociated ATPaseobtained from the control roots was apparently higher than theactivity obtained from Ca²⁺-starved roots. The amount of asolectinrequired for maximum activation of the partially lipid-depletedATPase prepared from control roots was much lower than thatprepared from Ca²⁺-starved roots. Reassociation of partiallylipid-depleted ATPase with asolectin produced higher ATPaseactivity than that with individual phospholipids. The activationof partially lipid-depleted ATPase prepared from control rootswith asolectin was not inhibited by addition of a sample preparedfrom Ca²⁺-starved roots. Thus, a decrease in the functionalassociation of ATPase with phospholipids might be one of thephysiological injuries in root cell membranes of cucumber causedby Ca²⁺ starvation. ¹Permanent address: Department of Horticulture, College of Agriculture,Chonnam National University, Chonnam 500, Korea. (Received February 23, 1988; Accepted August 18, 1988)     

Increase in Proton-Transport Activity of Tonoplast Vesicles as an Adaptive Response of Barley Roots to NaCl Stress

H⁺-Transport activity of the vesicles prepared from barley rootswas studied at the early phase after application of NaCl stress.The activity reached maximal level at 3 days after the treatmentwith 200 mM NaCl which moderately reduced the growth. This activityincrease could be suppressed in the presence of cycloheximideand actinomycin D. The properties of the membrane vesicles associated with H⁺-transportactivity prepared from both control and NaCl-stressed rootssuggested that it was of tonoplast origin based on the followingfindings: optimal pH at 7.5, strong inhibition by nitrate butnot by vanadate, and stimulation by chloride. The density gradient centrifugation of vesicles with DextranT70 did not show any detectable difference in the distributionpatterns of H⁺-transport activities between control and NaClstressedroots. Furthermore, K_m values for ATP of the H⁺-transport activityof vesicles prepared from control and NaCl-stressed roots werethe same. Therefore, H⁺-transport activity with properties similarto those of the control roots was increased by NaCl stress.The results are discussed in terms of an adaptive mechanismof barley against salt stress. ¹Permanent address: Department of Horticulture, College of Agriculture,Chonnam National University, Chonnam 500, Korea. (Received April 18, 1988; Accepted July 20, 1988)     

Expression and sequence analysis of a cDNA encoding the orotidine-5'-monophosphate decarboxylase domain from Ehrlich ascites uridylate synthase

Orotidine-5'-monophosphate decarboxylase (OD-Case) catalyzes the conversion of orotidine 5'-monophosphate to UMP. In mammals, ODCase is present as part of a bifunctional protein which also contains orotate phosphoribosyltransferase; the preceding enzyme in the de novo UMP biosynthetic pathway. We have isolated a plasmid (pMEJ) which contains a cDNA for the ODCase domain of UMP synthase. Insertion of this sequence into an Escherichia coli expression vector (pUC12) has allowed for the expression of ODCase and not orotate phosphoribosyltransferase in E. coli. The molecular weight of the expressed protein is 26,000-27,300 from immunoblot analysis which corresponds closely to the molecular weight of the ODCase domain (28,500) isolated by tryptic digestion of UMP synthase. We have sequenced the cDNA insert of pMEJ and deduced the amino acid sequence. The molecular weight of the ODCase domain calculated from the amino acid sequence in 28,654. Comparison of the deduced amino acid sequence from pMEJ with that for yeast ODCase (a monofunctional protein) demonstrated that 52% of the amino acids were identical when the two sequences are compared. Furthermore, several stretches of the amino acid sequence have 80% or greater absolute homology.     

Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds

A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.     

Secretion and localization of invertase and inulinase in recombinant Saccharomyces cerevisiae

Summary Secretion of invertase and inulinase produced by recombinant Saccharomyces cerevisiae cells were investigated under derepression conditions of GALI promoter. Secreted invertase mainly localized in the periplasmic space, but most of inulinase was found in the extracellular culture medium. This high level of extracellular secretion of inulinase was not dependent on the growth phase in which derepression of GALI promoter occurs. Our results indicate that the inulinase polypeptide itself may have a function for the protein secretion into the culture medium.