Northern Analysis Of Poly Research Articles

Alternative pre-mRNA splicing in Drosophila spliceosomal assembly factor RNP-4F during development

The 5′- and 3′-UTR regions in pre-mRNAs play a variety of roles in controlling eukaryotic gene expression, including translational modulation. Here we report the results of a systematic study of alternative splicing in rnp-4f, which encodes a Drosophila spliceosomal assembly factor. We show that most of the nine introns are constitutively spliced, but several patterns of alternative splicing are observed in two pre-mRNA regions including the 5′-UTR. Intron V is shown to be of recent evolutionary origin and is infrequently spliced, resulting in generation of an in-frame stop codon and a predicted truncated protein lacking a nuclear localization signal, so that alternative splicing regulates its subcellular localization. Intron 0, located in the 5′-UTR, is subject to three different splicing decisions in D. melanogaster. Northern analysis of poly(A +) mRNAs reveals two differently sized rnp-4f mRNA isoforms in this species. A switch in relative isoform abundance occurs during mid-embryo stages, when the larger isoform becomes more abundant. This isoform is shown to represent intron 0 unspliced mRNA, whereas the smaller transcript represents the product of alternative splicing. Comparative genomic analysis predicts that intron 0 is present in diverse Drosophila species. Intron 0 splicing results in loss of an evolutionarily conserved stem-loop constituting a potential cis-regulatory element at the 3′-splice site. A model is proposed for the role of this element both in 5′-UTR alternative splicing decisions and in RNP-4F translational modulation. Preliminary evidences in support of our model are discussed.

Molecular cloning and domain structure of chicken pyruvate carboxylase

Pyruvate carboxylase (PC) [EC 6.4.1.1] is a biotin-dependent carboxylase that catalyses the conversion of pyruvate to oxaloacetate. Here we have determined the complete nucleotide sequence encoding chicken PC (cPC) by screening a liver cDNA library, by RT-PCR of poly(A) + RNA, and by PCR of genomic DNA. The full-length transcript contains an open reading frame of 3537 nucleotides, including the stop codon, encoding a polypeptide of 1178 amino acids with M r of 127,262. The amino acid sequence of cPC shows approximately 77% identity to mammalian PC. Limited proteolysis of pure cPC with chymotrypsin yields a major stable 75 kDa C-terminal peptide, including the biotinyl domain and a minor, unstable 39 kDa N-terminal peptide. Northern analysis of poly(A) + RNA isolated from chicken liver has shown that cPC's mRNA is approximately 5 kb in length, including a very long 3 ′-untranslated region.

Gene Structure and Expression of the Targeting Subunit, RGL, of the Muscle-Specific Glycogen-Associated Type 1 Protein Phosphatase, PP1G

The type I phosphatase associated with glycogen, PP1G, plays an important role in glycogen metabolism. PP1G is targeted to glycogen by the RGL subunit, which regulates the function of the enzyme. We report the cloning and characterization of the gene as well as the pattern of expression of the RGL subunit from mouse. The gene covers more than 37 kb, is composed of four exons and three introns, and codes for a 1089 residue polypeptide with a calculated molecular weight of 121,000. The amino acid sequence has 60% identity with the human and rabbit RGL. The 5′ flanking region of the gene contains a TATA box, c-Myc sites, and a potential cAMP-responsive element. Muscle specific motifs, such as MyoD and MEF-2, were also found. The A-T rich 3′-UTR contained several polyadenylation signals, two associated with poly(A) downstream consensus motifs. ARE elements, which regulate mRNA stability, were dispersed throughout the 3′-UTR. Northern analysis of poly(A) mRNA from various murine tissues indicates a major transcript of 7.5 kb in skeletal muscle and heart. Western analysis demonstrates that RGL protein is present in skeletal and cardiac muscle from mouse, rat, and rabbit but not in L6 myoblasts, L6 myotubes, 3T3 L1 fibroblasts, 3T3 L1 or rat primary adipocytes, confirming that expression of the gene is specific to striated muscle. Analysis of skeletal muscle from rats made diabetic by streptozotocin treatment reveals that the level of RGL protein is the same as in control animals, indicating that expression is not regulated by insulin.

Identification, cellular distribution and potential function of the metalloprotease-disintegrin MDC9 in the kidney.

The complex interactions of glomerular and tubular epithelial cells with the basal laminae play a critical role in renal function. Disruption of these interactions has been widely implicated in glomerular diseases and acute renal failure. MDC are a large family of membrane-bound proteins containing metalloprotease, disintegrin (integrin interaction sites), and cysteine-rich domains. Little information is available concerning the presence of MDC in the kidney or their role in renal pathophysiology. Using degenerate PCR primers for the conserved metalloprotease and disintegrin domains of this protein family, cDNA templates from tubules, whole glomeruli, and glomerular epithelial cells (GEC) yielded a single, 195-bp product, which on sequence analysis corresponded to a region in the disintegrin domain of MDC9. Northern analysis of poly(A)+ RNA from tubules, whole glomeruli, and GEC revealed a 3.9-kb transcript, identical to that of mouse MDC9. Using antibodies generated against a 21-amino acid peptide present in the metalloprotease domain of MDC9, Western analysis of concanavalin A-enriched glomerular microsomal extracts demonstrated both processed (76 kD) and unprocessed (116 kD) forms of MDC9, which upon reduction changed to the corresponding 84- and 124-kD forms. Histochemical studies revealed a basolateral localization of intrinsic MDC9 protein in renal cortical tubule cells and glomerular visceral epithelial cells, which colocalized with the beta1 integrin chain. Expression of green fluorescence protein MDC9 chimeric constructs in GEC or polarized Madin-Darby canine kidney epithelial cells revealed a similar punctate basolateral surface localization. Transient overexpression of the soluble disintegrin domain-green fluorescence protein chimera in GEC led to dramatic changes in cellular morphology with rounding and detachment from cell monolayers. These studies document the presence of MDC9 in renal epithelial cells and suggest an important role for MDC9 in renal epithelial cellular interactions with the basal lamina and adjoining cells.

Expression of an extracellular calcium-sensing receptor in human and mouse bone marrow cells.

The cloning of a G protein-coupled, extracellular calcium (Ca2+e)-sensing receptor (CaR) from bovine parathyroid provided direct evidence that Ca2+e-sensing can occur through receptor-mediated activation of G proteins and their associated downstream regulators of cellular function. CaR transcripts and protein are present in various tissues of humans and other mammals that are involved in Ca2+e homeostasis, including parathyroid, kidney, and thyroidal C-cells. The present study was performed to determine whether bone marrow cells express the CaR, since cells within the marrow space could be exposed to substantial changes in Ca2+e related to bone turnover. Using DNA and RNA probes from the human parathyroid CaR cDNA, we identified CaR transcripts of 5.2 and approximately 4.0 kilobases by Northern analysis of poly(A+) RNA from low-density mononuclear cells isolated from whole human bone marrow that are putatively enriched in marrow progenitor cells, including bone cell precursors. In situ hybridization also identified CaR transcripts in the same cell preparations. Reverse transcription-polymerase chain reaction demonstrated > 99% nucleotide identity between transcripts from human bone marrow cells and the corresponding regions of the human CaR cDNA. Antisera specific for several different regions within the extracellular domain of the CaR were reactive with low-density human marrow cells that were either adherent or nonadherent to plastic. About one-third of the adherent, CaR-immunoreactive cells were also positive for alkaline phosphatase, a nonspecific marker of preosteoblasts, osteoblasts, and assorted cells of the colony-forming unit-fibroblast lineage. In addition, a substantial fraction (approximately 60%) of low density murine marrow cells cultured for 1 week at 4.8 mM Ca2+e expressed both CaR immunoreactivity and nonspecific esterase, an enzyme expressed by monocyte/macrophages and fibroblasts. Finally, erythroid precursors and megakaryocytes from murine marrow as well as blood platelets expressed abundant CaR immunoreactivity, while peripheral blood erythrocytes and most polymorphonuclear leukocytes did not. These studies indicate that the CaR is present in low-density mononuclear bone marrow cells as well as in cells of several hematopoietic lineages and could potentially play a role in controlling the function of various cell types within the marrow space.

Estrogen Receptor α and β Expression in Upper Gastrointestinal Tract with Regulation of Trefoil Factor Family 2 mRNA Levels in Ovariectomized Rats

This study compared expression of two estrogen receptor (ERα and ERβ) genes in the rat upper gastrointestinal tract and the effects of 17β-estradiol administration on gastric trefoil factor family (TFF) mRNA steady-state levels in ovariectomized rats. Estrogen receptor α and β cDNA fragments from fundic mucosa were cloned by reverse transcriptase polymerase chain reaction (RT–PCR) and sequenced. Both ER subtypes were detected in fundus, antrum and duodenum by RT–PCR. Northern analysis of poly(A) +mRNA from fundic mucosa showed that ERα mRNA is expressed as a single transcript at 6.5 kb and ERβ is expressed as multiple transcripts with major transcripts ranging from 1.1–4.7 kb. ERβ mRNA was expressed in greater abundance than ERα mRNA. Fundic TFF2 mRNA steady-state levels were increased by 17β-estradiol administration in ovariectomized rats with no significant change in TFF1 mRNA levels. These studies show expression of both ER subtypes in the rat upper gastrointestinal tract with regulation of TFF2 mRNA by 17β-estradiol. These results suggest that estrogens, probably acting via ERβ, have a direct role in regulating gastric physiology.

Endothelin receptor mRNAs in human fetal membranes, chorionic vessels, and decidua parietalis

Endothelin-1 (ET-1) is synthesized in avascular human amnion; and, immunoreactive ET-1 is present in amniotic fluid in concentrations 10 to 100 times those in blood. ET-1 acts, most commonly, in a local or paracrine manner; therefore, it is possible that amnion/amniotic fluid ET-1 acts on contiguous tissues, namely chorion laeve or placental surface (chorionic) vessels, or in an autocrine fashion on amnion cells. To address these possibilities, the levels of ET A and ET B receptor mRNAs were evaluated in amnion, chorion laeve, decidua parietalis, placenta, and chorionic vessel tissues. By Northern analysis of total RNA (20 μg), ET A and ET B receptor mRNAs were detected in decidua ( n=18), placenta ( n=14), and chorionic vessels ( n=13). In chorion laeve ( n=24), ET B receptor mRNA but not ET A receptor mRNA was detected by Northern analysis of total RNA. Northern analysis of chorion laeve poly(A) + mRNA (1.5–2.5 μg) revealed ET A receptor mRNA at low levels. Neither ET A nor ET B receptor mRNAs were detected in amnion tissue by Northern analysis of total RNA ( n=30; placental and reflected amnion from 15 pregnancies) or by Northern analysis of poly(A) + mRNA (1.5–2.5 μg). Moreover, there was no demonstrable dose-dependent effect of ET-1 on prostaglandin E 2 production or DNA synthesis in amnion epithelial cells in primary culture. The findings of this investigation are indicative that ET-1 in amniotic fluid or secreted from amnion may act in a paracrine fashion on chorion laeve by way of the ET B receptor and on chorionic vessels by way of ET A and ET B receptors.

Endothelins and human placental growth: A review

Endothelin-1 (ET-1) is synthesized in avascular human amnion; and, immunoreactive ET-1 is present in amniotic fluid in concentrations 10 to 100 times those in blood. ET-1 acts, most commonly, in a local or paracrine manner; therefore, it is possible that amnion/amniotic fluid ET-1 acts on contiguous tissues, namely chorion laeve or placental surface (chorionic) vessels, or in an autocrine fashion on amnion cells. To address these possibilities, the levels of ETA and ETB receptor mRNAs were evaluated in amnion, chorion laeve, decidua parietalis, placenta, and chorionic vessel tissues. By Northern analysis of total RNA (20 μg), ETA and ETB receptor mRNAs were detected in decidua (n=18), placenta (n=14), and chorionic vessels (n=13). In chorion laeve (n=24), ETB receptor mRNA but not ETA receptor mRNA was detected by Northern analysis of total RNA. Northern analysis of chorion laeve poly(A)+ mRNA (1.5–2.5 μg) revealed ETA receptor mRNA at low levels. Neither ETA nor ETB receptor mRNAs were detected in amnion tissue by Northern analysis of total RNA (n=30; placental and reflected amnion from 15 pregnancies) or by Northern analysis of poly(A)+ mRNA (1.5–2.5 μg). Moreover, there was no demonstrable dose-dependent effect of ET-1 on prostaglandin E2 production or DNA synthesis in amnion epithelial cells in primary culture. The findings of this investigation are indicative that ET-1 in amniotic fluid or secreted from amnion may act in a paracrine fashion on chorion laeve by way of the ETB receptor and on chorionic vessels by way of ETA and ETB receptors.

Identification of functional aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator in murine splenocytes

The objective of the present studies was to determine whether the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) protein are present and functional in B6C3F1 (C57BL/6 × C3H) mouse splenocytes. Northern analysis of poly (A) RNA isolated from splenocytes revealed transcripts of approximately 6.6 kb which hybridized to the AhR complementary DNA (cDNA) probe. Anti-AhR antibodies identified two major cytosolic forms of the AhR in splenocytes, approximately 95 and 104 kDa, corresponding to the codominately expressed Ahr b alleles in the B6C3F1 mice. Northern analysis utilizing an oligomer probe for ARNT identified three messenger RNA (mRNA) transcripts, approximately 5.6, 2.0, and 1.1 kb, in spleen which was consistent with the banding pattern observed in the B6C3F1 mouse liver. Western blotting confirmed the presence of the approximately 87 kDa ARNT protein in splenocytes. Protein quantitation by slot blot analysis demonstrated approximately 2.0-fold more AhR in liver than in splenocytes. Interestingly, ARNT was approximately 2.4-fold more abundant in splenocytes than in liver. Consistent with these results, comparison by quantitative reverse transcriptase-polymerase chain reaction analysis of AhR and ARNT transcripts in liver and splenocytes demonstrated approximately 2.3-fold more AhR transcripts in liver than in splenocytes and approximately 3.2-fold more ARNT transcripts in splenocytes than in liver. In addition, comparisons between AhR and ARNT transcripts isolated from the liver and splenocytes indicated a greater number of ARNT transcripts as compared with AhR in both preparations. TCDD treatment of splenocytes induced binding of the AhR nuclear complex to the dioxin-responsive enhancer (DRE) as detected by the electrophoretic mobility shift assay. These findings confirm that the AhR and ARNT are present in mouse splenocytes and are capable of binding to the DRE.

Cloning of wound-induced cytochrome P450 monooxygenases expressed in pea.

Cytochrome P450 monooxygenases (P450s) mediate a wide range of oxidative reactions involved in the biosynthesis of plant secondary metabolites including phenylpropanoids and phytoalexins. To investigate the regulation of these P450s in the phenylpropanoid biosynthetic pathway of pea (Pisum sativum), partial cDNAs representing four distinct P450s expressed in pea seedlings were cloned using a reverse transcription-polymerase chain reaction strategy. One of the corresponding full-length cDNA clones, designated CYP73A9, encodes pea trans-cinnamic acid 4-hydroxylase, which catalyzes the second core reaction in the phenylpropanoid pathway. As expected from its central role in the production of lignin precursors and defense compounds, northern analysis of poly(A)+ mRNA demonstrates that transcripts encoding CYP73A9 are induced appreciably within 3 h after wounding. A second cDNA clone, designated CYP82, encodes a novel P450, whose transcripts are also induced in response to wounding at approximately the same time as CYP73A9 transcripts. Despite the multitude of environmental stimuli known to induce expression of phenylpropanoid pathway enzymes, genomic DNA Southern analysis indicates that each of these P450s is encoded by a low copy number (possibly a single copy) gene family.

Trypanosoma brucei: Molecular Cloning of Homologues of Small GTP-Binding Proteins Involved in Vesicle Trafficking

Members of the rab subfamily of GTPases have been implicated as important components in vesicle trafficking in the eukaryotes. Individual rab proteins have a remarkable degree of specific subcellular localization. As a first approach to the study of these proteins in the protozoan parasite Trypanosoma brucei we have undertaken a survey of the rab subfamily using a strategy based on degenerate polymerase chain reaction utilizing the miniexon and the highly conserved WDTAGQE box which comprises part of the GTP-binding site. A number of T. brucei partial cDNAs were obtained from procyclic stage cDNA, and seven of these clones (designated rtb1, 3-7, 9) were clearly members of the rab family. Northern analysis of poly(A)-selected RNA indicates that rtb, 3, 4, and 7 are constitutively expressed at low levels in both life-stages of T. brucei. By Southern analysis of trypanosome genomic DNA and specific PCR from an isolated genomic clone, we show that two of the genes, RTB1 and RTB7, are adjacent or at least closely linked in the T. brucei genome, while the other five are dispersed. These data provide important molecular reagents for dissecting the unusual secretory pathway in this organism.

Expression Analysis, Genomic Structure, and Mapping to 7q31 of the Human Sperm Adhesion Molecule Gene SPAM1

During the course of systematic sequence tag analysis of clones isolated from an adult testis cDNA library, clones 296 and 576 were found to detect 71–74% sequence identity to the guinea pig sperm surface protein PH-20. This surface protein is involved in sperm–egg adhesion in the guinea pig. Nucleotide sequence for 1919 bp of human DNA from a series of overlapping cDNA clones isolated from a testis cDNA library confirmed the sequence identity within a 1527-bp open reading frame to be 71–74% to the guinea pig gene and the similarity to be 60% for the predicted protein of 509 amino acids. Southern blot analysis of human genomic DNA and DNA from somatic cell hybrids indicates that the gene (SPAM1) is unique and does not form part of a larger family and that it maps to chromosome 7. Fluorescencein situhybridization with yeast artificial chromosome (YAC) clones isolated from the CEPH megaYAC library has refined this localization to 7q31. PCR analysis of genomic DNA and YAC clone DNA has shown that the 1919 bp of the gene that has been cloned covers approximately 11 kb of genomic DNA and is encoded by at least 4 exons. Northern analysis of poly(A)+mRNA from a range of 16 human tissues has demonstrated that expression of the gene as a single 2.4-kb transcript is strictly limited to the testis.

ECM degradation by cultured human mesangial cells is mediated by a PA/plasmin/MMP-2 cascade

We examined the role of the plasminogen activator/plasmin system in extracellular matrix (ECM) degradation by human mesangial cells cultured on thin films of 125I-labeled ECM (Matrigel). ECM degradation (release of 125I into the medium) was dependent on exogenous plasminogen, proportional to the number of mesangial cells and amount of plasminogen added, and coincident with the appearance of plasmin in the medium. ECM degradation was completely blocked (P < 0.001) by two plasmin inhibitors, alpha-2-antiplasmin (40 micrograms/ml) and aprotinin (216 KIU/ml), and partially reduced (-33 +/- 1.8%, P < 0.01) by TIMP-1 (40 micrograms/ml), a specific inhibitor of matrix metalloproteinases. Zymography of medium obtained from cells cultured in the absence of plasminogen revealed the presence of latent matrix metalloproteinase-2 (MMP-2) which was converted to a lower molecular weight, active form in the presence of mesangial cells and plasminogen. Northern analysis of poly A+RNA prepared from cultured human mesangial cells revealed mRNA for tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and uPA receptor (uPAR). The presence of uPA protein in medium obtained from cultured human mesangial cells was demonstrated by Western blotting and ELISA which revealed a large molar excess of PAI-1 (1.2 +/- 0.1 x 10(-9) M) over uPA (1.2 +/- 0.1 x 10(-12) M) and tPA (0.19 +/- 0.04 x 10(-9) M). ECM degradation was reduced by a monoclonal antibody (MAb) against human tPA (-54 +/- 8.6%) or human uPA (-39 +/- 5.2%) compared to cells treated with identical amounts of non-specific monoclonal IgG (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

17 beta-Hydroxysteroid dehydrogenase type 2: chromosomal assignment and progestin regulation of gene expression in human endometrium.

The cDNAs for two separate human 17 beta-hydroxysteroid dehydrogenases (17 beta-HSD) have been isolated and sequenced. The well-studied human placental cytosolic 17 beta-HSD (also referred to as estradiol dehydrogenase) preferentially catalyzes the reduction of estrone to estradiol-17 beta and the reduction of the C-20-ketone of progesterone to 20 alpha-dihydroprogesterone. This isoform of the enzyme has been referred to as 17 beta-HSD type 1 and localized to chromosome 17. A second 17 beta-HSD isoform (referred to as type 2) is localized in the endoplasmic reticulum of human trophoblast and is characterized by the preferential oxidation of the C-17 beta-hydroxyl group of C18- and C19-steroids and the C-20 alpha-hydroxyl group of 20 alpha-dihydroprogesterone. In this study, we determined the chromosomal localization of human 17 beta-HSD type 2, the expression of this gene in human endometrium, and the tissue distribution of the mRNA. We found that the human 17 beta-HSD type 2 gene is localized on chromosome 16, 16q24. 17 beta-HSD type 2 mRNA (approximately 1.5 kb) was identified in human endometrial tissues by Northern analysis of total RNA (10 micrograms). The highest levels of 17 beta-HSD type 2 mRNA were found in endometrial tissues obtained during the mid- to late secretory phase of the ovarian cycle (i.e., during the time of high plasma levels of progesterone). 17 beta-HSD type 2 mRNA levels were much greater in glandular epithelium than in the stromal cells isolated from secretory phase endometrium. The levels of 17 beta-HSD type 2 mRNA in secretory phase endometrium were approximately one-tenth that in villous trophoblast tissue from human placenta. We did not detect 17 beta-HSD type 1 mRNA in endometrial tissue by Northern analysis of total (10 micrograms) RNA. These findings are consistent with the view that the progestin-regulated 17 beta-HSD of the glandular epithelium of the human endometrium is primarily, if not exclusively, the product of the 17 beta-HSD type 2 gene. 17 beta-HSD type 2 mRNA was present in human placenta, liver, and small intestine; much smaller amounts, barely detectable by Northern analysis of poly(A)+ RNA, were present in prostate, kidney, pancreas, and colon, but not in heart, brain, skeletal muscle, spleen, thymus, ovary, or testis.

CDNA cloning and characterization of a vitamin D3 hydroxylase-associated protein.

We previously reported the generation of monoclonal antibodies which immunoprecipitate a fraction of the total chick kidney 1,25-dihydroxyvitamin D3-24R-hydroxylase activity. These antibodies were used to screen a chick kidney lambda gt11 cDNA library resulting in the isolation of a full-length cDNA encoding a protein that is not the 1,25-dihydroxyvitamin D3-24R-hydroxylase but another protein we term the vitamin D3 hydroxylase-associated protein (VDHAP). The deduced amino acid sequence agreed with an NH2-terminal amino acid sequence from the isolated VDHAP. Gene and protein bank search did not identify homology to known sequences or functional domains in the VDHAP cDNA. VDHAP mRNA levels are not altered by conditions which either induce 1,25-dihydroxyvitamin D3-24R-hydroxylase activity (78-fold) or 25-hydroxyvitamin D3-1 alpha-hydroxylase activity (30-fold). Northern analysis of poly(A)+ RNA from chick tissues revealed VDHAP only in kidney. Cellular fractionation experiments demonstrated that VDHAP and the 25-hydroxyvitamin D3-1 alpha-hydroxylase are colocalized in the inner membrane of mitochondria. The VDHAP antibody immunoprecipitates 14% of the total 1,25-dihydroxyvitamin D3-24R-hydroxylase activity (7-fold over background) and immunoprecipitates 21% of the total 25-hydroxyvitamin D3-1 alpha-hydroxylase activity (2-fold over background). VDHAP is a novel chick kidney-specific inner membrane protein of mitochondria, which associates with a fraction of the 1,25-dihydroxyvitamin D3-24R-hydroxylase and 25-hydroxyvitamin D3-1 alpha-hydroxylase.

Porcine stem cell factor/c-kit ligand: its molecular cloning and localization within the uterus.

Stem cell factor (SCF), or c-kit ligand, is a multipotent growth factor that has been implicated in an important role in various aspects of animal development, including maintenance of the viability of primordial germ cells. A porcine SCF (pSCF) cDNA was generated from porcine uterine endometrial mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR), and its nucleotide sequence was determined. The 952-bp pSCF cDNA contained an open reading frame encoding 274 amino acids. The deduced amino acid sequence of pSCF is approximately 86%, 83%, and 82%, identical to human, rat, and mouse SCFs, respectively; and it contains the four conserved cysteine residues and Asn-linked glycosylation sites. One additional amino acid was identified in pSCF, Glu130, which is not in the human (hSCF), rat (rSCF), or mouse (mSCF) sequences. Northern analysis of poly(A)+ RNA obtained from Day 16 pregnant endometrium revealed a transcript of approximately 6.5 kb. The size of this transcript is consistent with the size of full-length SCF mRNA, and the occurrence of alternatively spliced pSCF mRNAs were not detected by RT-PCR/Southern hybridization analysis of endometrial and ovarian total cellular RNA (tcRNA). Porcine SCF mRNA has been localized by in situ hybridization in porcine endometrial stromal tissue. Pregnancy does not appear to be a prerequisite for pSCF mRNA expression in endometrial tissue since it was detectable in tissue and tcRNA obtained from pregnant and nonpregnant gilts. The biological significance of uterine pSCF expression is currently unclear, but it probably participates in intracellular communication within the uterus.

A splice variant of the [formula omitted] (NMDAR1) receptor

A splice variant of the NMDA receptor (NMDAR1) was discovered containing a deletion of 37 amino acids near the carboxyl tail and has been designated NMDAR1b. The 111 nucleotides corresponding to the deleted amino acid sequence were found in a separate exon bounded by consensus intron/exon junction sequences in rat genomic DNA. A partial restriction map of genomic DNA bounding this region placed the deleted exon approximately 600 base pairs (bp) downstream of the upstream exon. RT/PCR analysis of RNA from different brain regions showed that the deletion variant is more abundantly expressed in the brain stem and cerebellum while the full-length form is expressed more abundantly in the olfactory bulb, striatum, hippocampus, and cortex. Northern analysis of poly(A) + RNA from different brain regions with probes specific for the deleted exon (i.e., full-length form) and for the splice junction (deletion form) indicated approximately 4.4 kb transcripts. The probe for the deleted exon hybridized to transcripts in olfactory bulb, cortex, striatum, and hippocampus while the splice junction probe hybridized most strongly to transcripts in cerebellum. The results suggest an interesting rostral to caudal shift in the expression of splice variants of the NMDAR1 which may signify important functional differences in native forms of NMDA receptors.

Differential Expression of the Cellular Retinaldehyde-binding Protein in Bovine Ciliary Epithelium

Differential expression of the cellular retinaldehyde-binding protein (CRALBP), a likely component of the visual cycle, has been observed in the ocular ciliary epithelium, a bilayer of neurepithelial cells involved in the active transport of aqueous humor. Antibody probes raised to bovine ciliary epithelium were used to isolate a cDNA encoding CRALBP from a bovine ciliary epithelium cDNA expression library. Northern analysis of poly A + RNA isolated from three different regions of the neonate ciliary epithelium revealed a three-fold higher level of CRALBP expression in the most anterior portion of the pars plicata region relative to the posterior pars plana region. Western blot analysis using CRALBP-specific antibodies also demonstrated that the level of CRALBP expression was higher in the most anterior portion of the ciliary epithelium and gradually decreases toward the posterior region in both neonate and adult bovine eyes. Using indirect immunofluorescence and frozen-sectioned bovine ciliary epithelium, CRALBP was localized to the pigmented ciliary epithelial cell layer along the entire ciliary epithelium. In the nonpigmented ciliary epithelial cell layer, CRALBP was only detected in the pars plana region. Pigmented ciliary epithelial cells have also been cultured and CRALBP expression demonstrated for several continuous passages by Northern and Western analysis. These results raise questions regarding the physiological role of CRALBP in the ciliary epithelium and the involvement of the protein in defining regional and functional boundaries within this tissue.

A bone marrow stromal cell line is a source and target for platelet- derived growth factor

Platelet-derived growth factor (PDGF) stimulates multipotent and erythroid progenitors as well as stromal fibroblasts. Any of the three dimeric forms of PDGF (AA, AB, or BB) could potentially interact with these cells; however, the precise cellular origin of PDGF production in the bone marrow microenvironment is not known. In the present study, we found that medium conditioned by MBA-2, murine bone marrow-derived endothelial cells, contains PDGF activity that competes for [125I]PDGF binding to human foreskin fibroblasts and is mitogenic for these fibroblasts. Northern analysis of poly(A)+ RNA from MBA-2 shows the expression of both PDGF A-chain and B-chain mRNAs. Because cytokines such as transforming growth factor-beta (TGF-beta) regulate hematopoiesis and stimulate PDGF in certain mesenchymal cells, we determined whether TGF-beta influences PDGF secretion and gene expression in MBA-2. TGF-beta induced PDGF A-chain and B-chain mRNAs and the release of PDGF activity. Each of the three PDGF isoforms also stimulated DNA synthesis in MBA-2, but with different potency (BB = AB = AA). Ligand binding studies showed specific binding of labeled PDGF BB and, to a lesser extent, PDGF AA isoform, consistent with predominant expression of the PDGF-beta receptor in MBA-2. These data show that murine endothelial stromal cells release PDGF activity and respond to PDGF. Local production of PDGF in the marrow microenvironment may play an important role in regulating hematopoietic and stromal cell proliferation.

A Bone Marrow Stromal Cell Line Is a Source and Target for Platelet-Derived Growth Factor

AbstractPlatelet-derived growth factor (PDGF) stimulates multipotent and erythroid progenitors as well as stromal fibroblasts. Any of the three dimeric forms of PDGF (AA, AB, or BB) could potentially interact with these cells; however, the precise cellular origin of PDGF production in the bone marrow microenvironment is not known. In the present study, we found that medium conditioned by MBA-2, murine bone marrow-derived endothelial cells, contains PDGF activity that competes for [125I]PDGF binding to human foreskin fibroblasts and is mitogenic for these fibroblasts. Northern analysis of poly(A)+ RNA from MBA-2 shows the expression of both PDGF A-chain and B-chain mRNAs. Because cytokines such as transforming growth factor-β (TGF-β) regulate hematopoiesis and stimulate PDGF in certain mesenchymal cells, we determined whether TGF-β influences PDGF secretion and gene expression in MBA-2. TGF-β induced PDGF A-chain and B-chain mRNAs and the release of PDGF activity. Each of the three PDGF isoforms also stimulated DNA synthesis in MBA-2, but with different potency (BB > AB > AA). Ligand binding studies showed specific binding of labeled PDGF BB and, to a lesser extent, PDGF AA isoform, consistent with predominant expression of the PDGF-β receptor in MBA-2. These data show that murine endothelial stromal cells release PDGF activity and respond to PDGF. Local production of PDGF in the marrow microenvironment may play an important role in regulating hematopoietic and stromal cell proliferation.