Bovine alpha1,3-galactosyltransferase catalytic domain structure and its relationship with ABO histo-blood group and glycosphingolipid glycosyltransferases.

alpha1,3-galactosyltransferase (alpha3GalT, EC 2.4.1.151) is a Golgi-resident, type II transmembrane protein that transfers galactose from UDP-alpha-galactose to the terminal N:-acetyllactosamine unit of glycoconjugate glycans, producing the Galalpha1,3Galbeta1,4GlcNAc oligosaccharide structure present in most mammalian glycoproteins. Unlike most other mammals, humans and Old World primates do not possess alpha3GalT activity, which is relevant for the hyperacute rejection observed in pig-to-human xenotransplantation. The crystal structure of the catalytic domain of substrate-free bovine alpha3GalT, solved and refined to 2.3 A resolution, has a globular shape with an alpha/beta fold containing a narrow cleft on one face, and shares a UDP-binding domain (UBD) with the recently solved inverting glycosyltransferases. The substrate-bound complex, solved and refined to 2.5 A, allows the description of residues interacting directly with UDP-galactose. These structural data suggest that the strictly conserved residue E317 is likely to be the catalytic nucleophile involved in galactose transfer with retention of anomeric configuration as accomplished by this enzyme. Moreover, the alpha3GalT structure helps to identify amino acid residues that determine the specificities of the highly homologous ABO histo-blood group and glycosphingolipid glycosyltransferases.

Fine-structural cytochemical and immunocytochemical observations on nuclear bodies in the bovine 2-cell embryo.

Nuclear bodies occurring during the 2-cell stage of bovine embryos (obtained either by in vitro fertilisation of in vitro matured ovarian oocytes, or collection after fertilisation and cleavage in vivo) were studied using ultrastructural cytochemistry and immunocytochemistry to determine whether their occurrence may be linked with the onset of embryonic transcription. In addition, the species-specific ultrastructural features of the interchromatin structures of the 2-cell bovine embryo were displayed. Three different types of nuclear bodies were distinguished: (i) nucleolus precursor bodies (NPBs), (ii) loose bodies (LBs) and (iii) dense bodies (DBs). In order to determine their possible functional significance, we considered parallels between these three nuclear entities and interchromatin compartments reported in other cells. As detected by their preferential ribonucleoprotein staining, all types of nuclear bodies contained ribonucleoproteins. In contrast to the other types of nuclear bodies studied, NPBs contained argyrophilic proteins but in no case they did show morphological features of functional nucleoli. Both compact and vacuolated forms of NPBs were seen in both in vivo and in vitro embryos, sometimes simultaneously in the same nucleus. LBs and DBs reacted with antibodies to Sm antigen, indicating the presence of a group of nucleoplasmic, non-nucleolar small nuclear ribonucleoproteins (snRNPs). The immunoreactivity for Sm antigen was more intense and homogeneous in DBs than in LBs. DBs were seen in both categories of embryo. A possible kinship of DBs with the sphere organelle known from oocytes of different animal species or the prominent spherical inclusions of the early mouse embryo nuclei is suggested. The last type of intranuclear body, the LBs, showed a composite structure. Their granular component, occurring in clusters and displaying immunoreactivity for Sm antigen, was similar to interchromatin granules and was therefore named IG-like granules. Another component forming the LBs showed a much finer structure and a lower immunoreactivity with anti-Sm antibodies. We suggest that this amorphous component may be related to the IG-associated zone. All three types of intranuclear bodies were often seen close together, suggesting their possible mutual functional relationship. From these and other observations we conclude that the intranuclear bodies in 2-cell bovine embryos correspond, with the exception of the NPB, to similar structures/compartments supposed to accumulate inactive spliceosomal components in certain phases of somatic cell nucleus functions. Accordingly, the occurrence of such nuclear bodies does not represent cytological evidence for RNA synthesis. In contrast to this, an important morphological feature revealing the status of the bovine 2-cell embryo is the vacuolisation of the NPB.

A novel 14-base-pair regulatory element is essential for in vivo expression of murine beta4-galactosyltransferase-I in late pachytene spermatocytes and round spermatids.

During murine spermatogenesis, beginning in late pachytene spermatocytes, the beta4-galactosyltransferase-I (beta4GalT-I) gene is transcribed from a male germ cell-specific start site. We had shown previously that a 796-bp genomic fragment that flanks the germ cell start site and contains two putative CRE (cyclic AMP-responsive element)-like motifs directs correct male germ cell expression of the beta-galactosidase reporter gene in late pachytene spermatocytes and round spermatids of transgenic mice (N. L. Shaper, A. Harduin-Lepers, and J. H. Shaper, J. Biol. Chem. 269:25165-25171, 1994). We now report that in vivo expression of beta4GalT-I in developing male germ cells requires an essential and previously undescribed 14-bp regulatory element (5'-GCCGGTTTCCTAGA-3') that is distinct from the two CRE-like sequences. This cis element is located 16 bp upstream of the germ cell-specific start site and binds a male germ cell protein that we have termed TASS-1 (transcriptional activator in late pachytene spermatocytes and round spermatids 1). The presence of the Ets signature binding motif 5'-GGAA-3' on the bottom strand of the TASS-1 sequence (underlined sequence) suggests that TASS-1 is a novel member of the Ets family of transcription factors. Additional transgenic analyses established that an 87-bp genomic fragment containing the TASS-1 regulatory element was sufficient for correct germ cell-specific expression of the beta-galactosidase reporter gene. Furthermore, when the TASS-1 motif was mutated by transversion, within the context of the original 796-bp fragment, transgene expression was reduced 12- to 35-fold in vivo.

Determination of beta1,4-galactosyltransferase enzymatic activity by capillary electrophoresis and laser-induced fluorescence detection.

We have developed a nonradioactive method to assay UDP-Gal:beta-d-GlcNAcbeta1,4-galactosyltransferase (beta4GalT-I) enzymatic activity. Capillary electrophoresis combined with laser-induced fluorescence detection (CE-LIF) was employed to provide a baseline separation of FITC-conjugated O-GlcNAc-containing substrate peptides and galactose-capped product peptides, while at the same time allowing a level of detection in the low attomole range (10(-18)). The addition of 2 mM hexamethylene diamine to the borate-based capillary electrophoretic buffer modulated the electroosmotic flow, resulting in optimum separation of the glycopeptide product from reactant. beta4GalT-I activity was dependent upon the addition of both manganese and UDP-galactose. Using this assay, we show that two beta4GalT-I constructs, predicted to localize to different intracellular compartments, are enzymatically active when expressed in vitro using a rabbit reticulocyte transcription-translation system. The high sensitivity of product detection by CE-LIF in combination with in vitro transcription-translation is applicable to the facile determination of the enzymatic activity of other newly cloned glycosyltransferases.

The increased level of beta1,4-galactosyltransferase required for lactose biosynthesis is achieved in part by translational control.

beta1,4-Galactosyltransferase (beta4GalT-I) participates in both glycoconjugate biosynthesis (ubiquitous activity) and lactose biosynthesis (mammary gland-specific activity). In somatic tissues, transcription of the mammalian beta4GalT-I gene results in a 4.1-kb mRNA and a 3.9-kb mRNA as a consequence of initiation at two start sites separated by approximately 200 bp. In the mammary gland, coincident with the increased beta4GalT-I enzyme level ( approximately 50-fold) required for lactose biosynthesis, there is a switch from the 4.1-kb start site to the preferential use of the 3.9-kb start site, which is governed by a stronger tissue-restricted promoter. The use of the 3.9-kb start site results in a beta4GalT-I transcript in which the 5'- untranslated region (UTR) has been truncated from approximately 175 nt to approximately 28 nt. The 5'-UTR of the 4.1-kb transcript [UTR(4.1)] is predicted to contain extensive secondary structure, a feature previously shown to reduce translational efficiency of an mRNA. In contrast, the 5'-UTR of the 3.9-kb mRNA [UTR(3.9)] lacks extensive secondary structure; thus, this transcript is predicted to be more efficiently translated relative to the 4.1-kb mRNA. To test this prediction, constructs were assembled in which the respective 5'-UTRs were fused to the luciferase-coding sequence and enzyme levels were determined after translation in vitro and in vivo. The luciferase mRNA containing the truncated UTR(3.9) was translated more efficiently both in vitro (approximately 14-fold) and in vivo (3- to 5-fold) relative to the luciferase mRNA containing the UTR(4.1). Consequently, in addition to control at the transcriptional level, beta4GalT-I enzyme levels are further augmented in the lactating mammary gland as a result of translational control.

Stable expression of mammalian beta 1,4-galactosyltransferase extends the N-glycosylation pathway in insect cells.

An established lepidopteran insect cell line (Sf9) was cotransfected with expression plasmids encoding neomycin phosphotransferase and bovine beta 1,4-galactosyltransferase. Neomycin-resistant transformants were selected, assayed for beta 1,4-galactosyltransferase activity, and the transformant with the highest level of enzymatic activity was characterized. Southern blots indicated that this transformed Sf9 cell derivative contained multiple copies of the galactosyltransferase-encoding expression plasmid integrated at a single site in its genome. One-step growth curves showed that these cells supported normal levels of baculovirus replication. Baculovirus infection of the transformed cells stimulated beta 1,4-galactosyltransferase activity almost 5-fold by 12 h postinfection. This was followed by a gradual decline in activity, but the infected cells still had about as much activity as uninfected controls as late as 48 h after infection and they were able to produce a beta 1,4-galactosylated virion glycoprotein during infection. Infection of the transformed cells with a conventional recombinant baculovirus expression vector encoding human tissue plasminogen activator also resulted in the production of a galactosylated end-product. These results demonstrate that stable transformation can be used to add a functional mammalian glycosyltransferase to lepidopteran insect cells and extend their N-glycosylation pathway. Furthermore, stably-transformed insect cells can be used as modified hosts for conventional baculovirus expression vectors to produce foreign glycoproteins with "mammalianized" glycans which more closely resemble those produced by higher eucaryotes.

The expanding beta 4-galactosyltransferase gene family: messages from the databanks.

From a systematic search of the UniGene and dbEST databanks, using human beta 4-galactosyltransferase (beta 4GalT-I), which is recognized to function in lactose biosynthesis, as the query sequence, we have identified five additional gene family members denoted as beta 4GalT-II, -III, -IV, -V, and -VI. Complementary DNA clones containing the complete coding regions for each of the five human homologs were obtained or generated by a PCR-based strategy (RACE) and sequenced. Relative to beta 4GalT-I, the percent sequence identity at the amino acid level between the individual family members, ranges from 33% (beta 4GalT-VI) to 55% (beta 4GalT-II). The highest sequence identity between any of the homologs is between beta 4GalT-V and beta 4GalT-VI (68%). beta 4GalT-II is the ortholog of the chicken beta 4GalT-II gene, which has been demonstrated to encode an alpha-lactalbumin responsive beta 4-galactosyltransferase (Shaper et al., J. Biol. Chem., 272, 31389-31399, 1997). As established by Northern analysis, beta 4GalT-II and -IV show the most restricted pattern of tissue expression. High steady state levels of beta 4GalT-II mRNA are seen only in fetal brain and adult heart, muscle, and pancreas; relatively high levels of beta 4GalT-VI mRNA are seen only in adult brain. When the corresponding mouse EST clone for each of the beta 4GalT family members was used as the hybridization probe for Northern analysis of murine mammary tissue, transcription of only the beta 4GalT-I gene could be detected in the lactating mammary gland. These observations support the conclusion that among the six known beta 4GalT family members in the mammalian genome, that have been generated through multiple gene duplication events of an ancestral gene(s), only the beta 4GalT-I ancestral lineage was recruited for lactose biosynthesis during the evolution of mammals.

Murine sperm-zona binding, a fucosyl residue is required for a high affinity sperm-binding ligand. A second site on sperm binds a nonfucosylated, beta-galactosyl-capped oligosaccharide.

An essential initial step in murine fertilization is the binding of acrosome-intact sperm to specific O-linked oligosaccharides on zona pellucida glycoprotein 3. While there is agreement on the primary role of O-linked glycans in this process, there is a lack of consensus on both the terminal monosaccharide(s) required for a functional sperm binding site and the corresponding protein on the sperm cell surface that recognizes this ligand. Much current debate centers on an essential role for either a terminal N-acetylglucosaminyl or, alternatively, a terminal alpha-galactosyl residue. To gain insight into the terminal saccharides required to form a functional sperm-binding ligand, dose-response curves were generated for a series of related tri- and tetrasaccharides to evaluate their relative effectiveness to competitively inhibit the in vitro binding of murine sperm to zona pellucida-enclosed eggs. A GlcNAc-capped trisaccharide, GlcNAc beta 1,4GlcNAc beta 1,4GlcNAc,was inactive (1-72 microM range). In contrast, a beta 4-galactosyl-capped trisaccharide (Gal beta 1,4GlcNAc beta 1, 4GlcNAc) and an alpha 3-galactosyl-capped trisaccharide (Gal alpha 1,3Gal beta 1,4 GlcNAc) inhibited sperm-zona binding with low or moderate affinity (ED50 = 42 microM and 5.3 microM, respectively). The addition of an alpha 3-fucosyl residue to each of these two competitive inhibitors, forming Gal beta 1,4[Fuc alpha 1,3] GlcNAc beta 1,4GlcNAc or Gal alpha 1,3Gal beta 1, 4[Fuc alpha 1,3]Glc NAc, resulted in ligands with 85- and 12-fold higher affinities for sperm, respectively (ED50 = 500 and 430 nM). Thus, the presence of a fucosyl residue appears to be obligatory for an oligosaccharide to bind sperm with high affinity. Last, mixing experiments with pairs of competitive inhibitors suggest that murine sperm-zona binding is mediated by two independent oligosaccharide-binding sites on sperm. The first (apparently high affinity) site binds both the alpha 3-galactosyl-capped trisaccharide and the two fucosylated tetrasaccharides. The second (apparently low affinity) site binds a nonfucosylated beta-galactosyl-capped trisaccharide.

Beta1,4-galactosyltransferase and lactose biosynthesis: recruitment of a housekeeping gene from the nonmammalian vertebrate gene pool for a mammary gland specific function.

Beta1,4-galactosyltransferase (beta4GalT-I) is a constitutively expressed trans-Golgi enzyme, widely distributed in vertebrates, which synthesizes the beta4-N-acetyllactosamine structure commonly found in glycoconjugates. In mammals beta4GalT-I has been recruited for a second biosynthetic function, the production of lactose; this function takes place exclusively in the lactating mammary gland. In preparation for lactose biosynthesis, beta4GalT-I enzyme levels are increased significantly. We show that mammals have evolved a two-step mechanism to achieve this increase. In step one there is a switch to the use of a second transcriptional start site, regulated by a stronger, mammary gland-restricted promoter. The transcript produced is distinguished from its housekeeping counterpart by the absence of approximately 180 nt of 5'-untranslated sequence. In step two, this truncated transcript is translated more efficiently, relative to the major transcript expressed in all other somatic tissues.

The chicken genome contains two functional nonallelic beta1,4-galactosyltransferase genes. Chromosomal assignment to syntenic regions tracks fate of the two gene lineages in the human genome.

Two distinct but related groups of cDNA clones, CKbeta4GT-I and CKbeta4GT-II, have been isolated by screening a chicken hepatoma cDNA library with a bovine beta1,4-galactosyltransferase (beta4GT) cDNA clone. CKbeta4GT-I is predicted to encode a type II transmembrane glycoprotein of 41 kDa with one consensus site for N-linked glycosylation. CKbeta4GT-II is predicted to encode a type II transmembrane glycoprotein of 43 kDa with five potential N-linked glycosylation sites. At the amino acid level, the coding regions of CKbeta4GT-I and CKbeta4GT-II are 52% identical to each other and 62 and 49% identical, respectively, to bovine beta4GT. Despite this divergence in amino acid sequence, high levels of expression of each cDNA in Trichoplusia ni insect cells demonstrate that both CKbeta4GT-I and CKbeta4GT-II encode an alpha-lactalbumin-responsive, UDP-galactose:N-acetylglucosamine beta4-galactosyltransferase. An analysis of CKbeta4GT-I and CKbeta4GT-II genomic clones established that the intron positions within the coding region are conserved when compared with each other, and these positions are identical to the mouse and human beta4GT genes. Thus CKbeta4GT-I and CKbeta4GT-II are the result of the duplication of an ancestral gene and subsequent divergence. CKbeta4GT-I maps to chicken chromosome Z in a region of conserved synteny with the centromeric region of mouse chromosome 4 and human chromosome 9p, where beta4-galactosyltransferase (EC 2.4.1.38) had previously been mapped. Consequently, during the evolution of mammals, it is the CKbeta4GT-I gene lineage that has been recruited for the biosynthesis of lactose. CKbeta4GT-II maps to a region of chicken chromosome 8 that exhibits conserved synteny with human chromosome 1p. An inspection of the current human gene map of expressed sequence tags reveals that there is a gene noted to be highly similar to beta4GT located in this syntenic region on human chromosome 1p. Because both the CKbeta4GT-I and CKbeta4GT-II gene lineages are detectable in mammals, duplication of the ancestral beta4-galactosyltransferase gene occurred over 250 million years ago in an ancestral species common to both mammals and birds.

Transcriptional regulation of murine beta1,4-galactosyltransferase in somatic cells. Analysis of a gene that serves both a housekeeping and a mammary gland-specific function.

beta1,4-Galactosyltransferase (beta4-GT) is a constitutively expressed enzyme that synthesizes the beta4-N-acetyllactosamine structure in glycoconjugates. In mammals, beta4-GT has been recruited for a second biosynthetic function, the production of lactose which occurs exclusively in the lactating mammary gland. In somatic tissues, the murine beta4-GT gene specifies two mRNAs of 4. 1 and 3.9 kilobases (kb), as a consequence of initiation at two different start sites approximately 200 base pairs apart. We have proposed that the region upstream of the 4.1-kb start site functions as a housekeeping promoter, while the region adjacent to the 3.9-kb start site functions primarily as a mammary gland-specific promoter (Harduin-Lepers, A., Shaper, J. H., and Shaper, N. L. (1993) J. Biol. Chem. 268, 14348-14359). Using DNase I footprinting and electrophoretic mobility shift assays, we show that the region immediately upstream of the 4.1-kb start site is occupied mainly by the ubiquitous factor Sp1. In contrast, the region adjacent to the 3.9-kb start site is bound by multiple proteins which include the tissue-restricted factor AP2, a mammary gland-specific form of CTF/NF1, Sp1, as well as a candidate negative regulatory factor that represses transcription from the 3.9-kb start site. These data experimentally support our conclusion that the 3.9-kb start site has been introduced into the mammalian beta4-GT gene to accommodate the recruited role of beta4-GT in lactose biosynthesis.

The gene encoding murine alpha 1,3-galactosyltransferase is expressed in female germ cells but not in male germ cells.

An essential step in murine fertilization is the binding of acrosome-intact sperm to specific O-linked glycans on zona pellucida glycoprotein 3 (ZP3). While there is agreement on the primary role of O-linked glycans in sperm-ZP3 binding, there is a striking lack of consensus on both the terminal monosaccharide(s) required for a functional binding site and the cognate protein on the sperm cell surface that recognizes this glycan. Much current debate centers on the essential role of nonreducing terminal N-acetyl-glucosaminyl or alternatively, alpha-galactosyl residues, to form a functional sperm binding ligand. Relevant to this debate, we demonstrated that alpha 1,3-galactosyltransferase (alpha 3-GT), which adds nonreducing terminal alpha-galactosyl residues to glycans, is not expressed in murine spermatocytes or spermatids. The objectives of this study were to determine whether alpha 3-GT is expressed in female germ cells and to compare the pattern of expression of two other terminal glycosyltransferases, beta 1,4-galactosyltransferase (beta 4-GT) and alpha 2,6-sialyltransferase (alpha 6-ST), between male and female germ cells. Total RNA was isolated from growing oocytes obtained from 15-day-old animals, fully grown oocytes, and eggs as well as spermatogonia, spermatocytes, and spermatids. The presence of alpha 3-GT, beta 4-GT, and alpha 6-ST mRNAs was analyzed by an RT-PCR-based assay. Our data demonstrate that the alpha 3-GT gene is expressed in female germ cells, but not in male germ cells. In contrast, both beta 4-GT and alpha 6-ST are expressed during oogenesis and spermatogenesis. This differential expression of alpha 3-GT in female germ cells is consistent with the model of sperm-egg binding in which a nonreducing terminal alpha-galactosyl residue is required for a functional determinant on ZP3 and with our hypothesis that the biological significance for the suppression of alpha 3-GT expression in male germ cells is to prevent sperm-sperm aggregation.

Male germ cell expression of murine beta 4-galactosyltransferase. A 796-base pair genomic region, containing two cAMP-responsive element (CRE)-like elements, mediates male germ cell-specific expression in transgenic mice.

In murine somatic cells, transcription of the single gene encoding beta 4-galactosyltransferase results in two transcripts of 4.1 and 3.9 kilobases (kb), as a consequence of the use of two transcriptional start sites that are located on exon one separated by 200 base pairs (bp). In early male germ cell development, spermatogonia use only the 4.1-kb start site to yield a transcript that is identical to its somatic cell counterpart. As these cells enter meiosis, there is a switch from the use of this somatic cell start site to the exclusive use, beginning in pachytene spermatocytes, of a male germ cell-specific start site. Germ cell-specific transcripts are distinguished from their somatic counterparts by an additional approximately 560 nucleotides of 5'-untranslated sequence that is located immediately upstream and contiguous with the transcriptional start site defined for the 4.1-kb mRNA (Harduin-Lepers, A., Shaper, N.L., Mahoney, J.A., and Shaper, J.H. (1992) Glycobiology 2, 361-368). This observation predicts the use of a different upstream male germ cell-specific promoter. In this study we show that a 796-bp fragment containing 543 bp of genomic sequence upstream of the germ cell specific transcriptional start site and 253 bp of flanking downstream sequence, directs expression of the reporter gene, beta-galactosidase, exclusively to the pachytene spermatocytes and round spermatids of transgenic mice. This pattern of cell type-specific expression of the transgene is comparable with that of the endogenous beta 4-galactosyltransferase gene.

Characterization of two cis-regulatory regions in the murine beta 1,4-galactosyltransferase gene. Evidence for a negative regulatory element that controls initiation at the proximal site.

The beta 1,4-galactosyltransferase (beta 1,4-GT) gene is unusual in that it specifies two mRNAs in somatic cells of 3.9 and 4.1 kilobases (kb). These two transcripts arise as a consequence of initiation at two different sets of start sites that are separated by approximately 200 base pairs. Translation of each mRNA results in the predicted synthesis of two related protein isoforms that differ only in the length of their NH2-terminal cytoplasmic domain (Russo, R.N., Shaper, N. L., and Shaper, J.H. (1990) J. Biol. Chem. 265, 3324-3331). In this study we show that the cellular requirements for beta 1,4-GT correlate with the transcriptional start site used. In cells and tissues that express low transcript levels, the 4.1-kb transcriptional start site is apparently used exclusively. Increased transcription from the 4.1-kb start site plus low levels of transcription from the 3.9-kb start site result in the intermediate beta 1,4-GT transcript levels that are found in almost all somatic cell types. However, in mid- to late pregnant and lactating mammary glands very high transcript levels are observed, which correlate with the predominant use of the 3.9-kb transcriptional start site. To identify the cis-acting elements that regulate the use of the two transcriptional start sites, we constructed a series of beta 1,4-GT/CAT hybrids and carried out transient transfection assays using mouse L cells and Drosophila SL2 cells. These studies have delineated both a distal and proximal regulatory region just upstream of the 4.1- and 3.9-kb transcriptional start sites, respectively. In addition, a negative cis-acting regulatory region was identified that represses transcription from the proximal site. These results suggest a model of transcriptional regulation in which the distal region functions as a housekeeping promoter while the proximal region functions as a mammary cell-specific promoter. Differential initiation from the two promoters is a mechanism for regulation of beta 1,4-GT enzyme levels. The predictions from this model are consistent with the conclusion that both protein isoforms are functionally equivalent resident trans-Golgi membrane-bound enzymes.

Immunocytochemical localization of beta 1,4 galactosyltransferase in epithelial cells from bovine tissues using monoclonal antibodies.

Post-embedding immunocytochemistry was employed to investigate the distribution of UDP-galactose:N-acetylglucosamine galactosyltransferase (beta 1,4-GT) in epithelial cells from various bovine organs. Several well characterized monoclonal antibodies previously demonstrated to recognize distinct polypeptide epitopes within the primary structure of beta 1,4-GT were applied to thin sections from tissues embedded in Lowicryl K4M, followed by the protein A-gold technique. Immunoreactivity was observed in the Golgi apparatus of epithelial cells from intestine, thymus and trachea. No immunoreactivity was observed in other intracellular structures, including rough endoplasmic reticulum, nuclear envelope and goblet cell mucus droplets. Within the Golgi apparatus, the staining was restricted to several cisternae in the trans region, with most portions of the trans-Golgi network appearing unlabelled. However, in thymic epithelial-reticular cells trans-Golgi network portions resembling classical GERL elements were stained by the antibodies. Thus, although immunoreactivity was subcompartmentalized within the Golgi apparatus in all epithelial cell types examined, the extent of staining within the trans-Golgi network was variable. Immunoreactivity was not detected at the plasma membrane (ecto-galactosyl-transferase), except in the case of a subpopulation of tracheal cells that resemble brush cells. These results suggest that in the epithelial cells examined, the subcompartmental distribution of beta 1,4-GT within the Golgi apparatus is maintained across different types of epithelial cell organization. Moreover, no evidence for a general epithelial cell ecto-galactosyltransferase could be discerned with these reagents.

Murine beta 1,4-galactosyltransferase: round spermatid transcripts are characterized by an extended 5'-untranslated region.

We have previously shown that the expression of the gene encoding murine beta 1,4-galactosyltransferase (beta 1,4-GT, UDP-galactose:N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D galactosyltransferase, EC 2.4.1.38) is fundamentally different between somatic and male germ cells (Shaper et al., 1990b). In somatic cells, two transcripts of 3.9 kb and 4.1 kb are produced. In contrast, in spermatogonia only the 4.1 kb transcript is expressed. Maturation of spermatogonia to pachytene spermatocytes is accompanied by reduced expression of the 4.1 kb transcript to barely detectable levels. Continued differentiation to haploid round spermatids is coincident with renewed expression in which the 4.1 kb transcript is replaced by two truncated transcripts of 2.9 and 3.1 kb. In this study, we report the characterization of a full-length beta 1,4-GT cDNA clone from a murine round spermatid library that corresponds to the 2.9 kb transcript. This transcript encodes the same open reading frame as the 4.1 kb transcript, but utilizes alternative poly(A) signals embedded within the long 3'-untranslated region of the somatic transcript. Based on sequence analysis, together with primer extension and S1 nuclease protection experiments, both the 2.9 and the 3.1 kb round spermatid beta 1,4-GT transcripts are distinguished by the presence of an additional 5'-untranslated sequence of approximately 560 bp that is absent in premeiotic germ cells and somatic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that rodent epididymal sperm contain the Mr approximately 94,000 glucocorticoid receptor but lack the Mr approximately 90,000 heat shock protein.

Monoclonal antibodies directed against four different polypeptide epitopes on the Mr approximately 94,000 steroid-binding subunit of the rat liver cytosolic glucocorticoid receptor (GcR) were used to probe Western blots of epididymal spermatozoa from rats and mice. Two sperm polypeptides with apparent molecular weights of 94,000 (indistinguishable in size from the liver GcR subunit) and 150,000 reacted with these antibodies. Other polypeptides that are present in a wide variety of somatic cells [lamin-A, -B, and -C; topoisomerase-I; poly(ADP-ribose) polymerase; the 62-kilodalton internal nuclear matrix protein; the nucleolar protein B23; and histone H1] could not be detected in these preparations of spermatozoa, thus appearing to rule out contamination by somatic cells. Rat and mouse pachytene spermatocytes and round spermatids contained much lower amounts of the Mr approximately 94,000 and 150,000 polypeptides. These results suggested that the steroid-binding subunit of the GcR might be accumulated late in spermatogenesis. Consistent with this view, a 6-kilobase mRNA (identical in size to a mRNA detected in mouse somatic cell lines) was detected when Northern blots of mouse round spermatid RNA were probed with a cDNA to the steroid-binding GcR subunit. Although the results described above suggest the presence of GcR in rodent sperm, high affinity binding of glucocorticoids to epididymal sperm could not be detected in a whole cell binding assay. Further analysis revealed that the Mr approximately 90,000 heat shock protein (hsp90), a component reportedly required for high affinity ligand binding to the GcR, was present in early germ cells, but absent from rodent epididymal sperm. These results suggest that the Mr approximately 94,000 steroid-binding subunit of the GcR and an immunologically related Mr approximately 150,000 polypeptide are specifically accumulated during the later stages of rodent spermatogenesis, but are not assembled into receptor complexes capable of binding steroid. In addition, these results support the view that hsp90 is required for high affinity binding of glucocorticoids to the Mr approximately 94,000 GcR subunit in intact cells.