cDNA In Mammalian Cells Research Articles

Biochemical and Kinetic Characterization of Angiotensin I Converting Enzymes (ACE) with Site‐Directed Amino Acid Changes: Interactions with Its Specific Inhibitors

ACE is responsible for generating the vasoconstrictor peptide angiotensin II of the Renin Angiotensin Aldosterone System, and blockage of this system with the use of ACE inhibitors and antagonists of angiotensin II receptors are strategies for treatment of hypertension. Studies demonstrated that the use of site-directed mutagenesis and the construction of chimeric enzymes can assist in the identification of the amino acids responsible for the interaction of enzyme/substrate or enzyme-inhibitor complexes. We investigated the role of amino acid residues targets located in the N-domain of ACE, Ala361, Thr378 and Phe467, strong candidates for the interaction of the enzyme with its inhibitors. The enzyme mutants were expressed in CHO cells, purified and characterized by enzymatic activity using specific substrates, inhibition assays with captopril, lisinopril and enalapril. After plasmid expression of wild type ACE (WT) cDNA in mammalian cells, PCR was performed to insert mutations 1 to 9 (M1 to M9), followed by electrophoresis, bacterial transfection, amplification of plasmids and sequencing. Only mutations M4 (A361W), M7 (T378A) and M8 (F437A) showed satisfactory results. M4, M7, M8 and WT were purified by a Superdex 200 column coupled to the FPLC system. The purified enzymes were characterized biochemically through enzymatic assays using the specific substrates for ACE, Hippuryl-His-Leu (HHL) and Z-Phe-His-Leu (z-FHL). WT, M4 and M7 showed high specific activity values. Analyzing the catalytic efficiency (kcat/KM), we observed that mutant enzymes better hydrolyzed the substrate z-FHL when compared to HHL. M7 showed higher specificity (0.39 mM) and catalytic velocity (1.02 s-1) for the substrate z-FHL. The one with the best specificity for the HHL substrate (1.35 mM) and catalytic velocity (0.54 s-1) was M4, compared to the WT. All enzymes showed an optimum pH around 8.0 with both substrates. Captopril inhibited the enzymes most efficiently using HHL as substrate, while Lisinopril was the one that best inhibited the enzymes using z-FHL. It suggests that there is a negative cooperation between the ACE catalytic sites by the substrate. For both inhibitors and substrates, the best inhibition occurred with M7, suggesting that this mutation may have altered the interaction of the enzyme active site. The results obtained in this work can lead to a better understanding of the importance of these amino acids residues for the activity/functionality of the enzyme, seeking the identification of new molecules that can inhibit ACE.

Abstract 011: Identification and Electrophysiological Characterization of a Novel Somatic Mutation (insT149KCNJ5) of the Potassium Channel Kir3.4 (KCNJ5)

Context: Understanding the function of the Kir3.4 (KCNJ5 gene) potassium channel through characterization of occurring novel mutations is key for dissecting the mechanism(s) of autonomous aldosterone secretion in primary aldosteronism. Objective: To identify novel KCNJ5 channel mutations and functionally characterize them in a large database of patients with aldosterone-producing adenomas (APA). Methods: We sequenced the APA and germinal DNA of 195 consecutive patients, diagnosed with the four corners criteria of the PAPY study. Among the 24.6% (48/195) APA patients who showed somatic KCNJ5 mutations we discovered a novel c.446insAAC insertion resulting in the mutant protein KCNJ5-insT149 in a patient with severe drug-resistant hypertension. The mutated cDNA generated by site-directed mutagenesis was transfected along with KCNJ3 cDNA in mammalian cells. 17α-hydroxylase, CYP11B1, and CYP11B2 were immunochemically localized in the excised adrenal gland. Whole-cell patch clamp recordings, CYP11B2 mRNA, aldosterone and intracellular Ca2+ measurement (Fura-2), and molecular modeling were performed to characterize the KCNJ5-insT149 mutation. Results: The patient’s high blood pressure was long-term cured; his LVMI fell from 168 to 106 g/m2 after 2 years follow-up. Compared to wild type and mock-transfected HAC15 adrenocortical cells, those expressing the mutant KCNJ5 showed increased CYP11B2 expression (expression fold change: 2.9±0.3, p<0.05 vs mock transfected cells) and aldosterone secretion (260 pg/μg RNA, p<0.05 vs mock cells). The HEK293 cells expressing the mutated KCNJ5-insT149 channel exhibited a strong Na+ inward current, and a substantial rise in intracellular Ca2+. The L-type Ca2+ channel blocker verapamil [10 μM] inhibited by 50% the pathological Na+ inward current, while both the Na+/Ca2+ exchanger blocker KB-R7943 [10 μM] and the removal of extracellular Na+ abolished it. Conclusions: We identified a novel mutation of the Kir3.4 channelopathy located after the pore α-helix preceding the selectivity filter, which causes pathological Na+ permeability, membrane depolarization, raised cytosolic Ca2+, and constitutive hypersecretion of aldosterone resulting in ensuing pseudo resistant hypertension.

Characterization of functional mannose receptor in a continuous hybridoma cell line

BackgroundThe mannose receptor is the best described member of the type I transmembrane C-type lectins; however much remains unanswered about the biology of the receptor. One difficulty has been the inability to consistently express high levels of a functional full length mannose receptor cDNA in mammalian cells. Another difficulty has been the lack of a human macrophage cell line expressing a fully functional receptor. Commonly used human macrophage cell lines such as U937, THP-1, Mono-Mac and HL60 do not express the mannose receptor. We have developed a macrophage hybridoma cell line (43MR cells) created by fusion of U937 cells with primary human monocyte-derived macrophages, resulting in a non-adherent cell line expressing several properties of primary macrophages. The purpose of this study was to identify and select mannose receptor-expressing cells using fluorescence-activated cell sorting and to characterize the expression and function of the receptor.ResultsIn the current study we show that the mannose receptor found on this novel cell has endocytic characteristics consistent with and similar to the mannose receptor found on the surface of monocyte-derived human macrophages and rat bone marrow-derived macrophages. In addition, we demonstrate that these cells engage and internalize pathogen particles such as S. aureus and C. albicans. We further establish the transfectability of these cells via the introduction of a plasmid expressing influenza A hemagglutinin.ConclusionsThe 43MR cell line represents the first naturally expressed MR-positive cell line derived from a human macrophage background. This cell line provides an important cell model for other researchers for the study of human MR biology and host-pathogen interactions.

A catecholamine transporter from the human parasite Schistosoma mansoni with low affinity for psychostimulants

The trematode Schistosoma mansoni is the primary cause of schistosomiasis, a devastating neglected tropical disease that affects 200 million individuals. Identifying novel therapeutic targets for the treatment of schistosomiasis is therefore of great public interest. The catecholamines norepinephrine (NE) and dopamine (DA) are essential for the survival of the parasite as they cause muscular relaxation and a lengthening in the parasite and thereby control movement.Here we characterize a novel dopamine/norepinephrine transporter (SmDAT) gene transcript, from S. mansoni. The SmDAT is expressed in the adult form and in the sporocyst form (infected snails) of the parasite, and also in the egg and miracidium stage. It is absent in the cercariae stage but curiously a transcript missing the exon encoding transmembrane domain 8 was identified in this stage.Heterologous expression of the cDNA in mammalian cells resulted in saturable, dopamine transport activity with an apparent affinity for dopamine comparable to that of the human dopamine transporter. Efflux experiments reveal notably higher substrate selectivity compared with its mammalian counterparts as amphetamine is a much less potent efflux elicitor against SmDAT compared to the human DAT. Pharmacological characterization of the SmDAT revealed that most human DAT inhibitors including psychostimulants such as cocaine were significantly less potent in inhibiting SmDAT. Like DATs from other simpler organisms the pharmacology for SmDAT was more similar to the human norepinephrine transporter. We were not able to identify other dopamine transporting carriers within the completed parasite genome and we hypothesize that the SmDAT is the only catecholamine transporter in the parasite and could be responsible for not only clearing DA but also NE.

Dominant inhibition of Fas ligand-mediated apoptosis due to a heterozygous mutation associated with autoimmune lymphoproliferative syndrome (ALPS) Type Ib

Background:Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of lymphocyte homeostasis and immunological tolerance due primarily to genetic defects in Fas (CD95/APO-1; TNFRSF6), a cell surface receptor that regulates apoptosis and its signaling apparatus.Methods:Fas ligand gene mutations from ALPS patients were identified through cDNA and genomic DNA sequencing. Molecular and biochemical assessment of these mutant Fas ligand proteins were carried out by expressing the mutant FasL cDNA in mammalian cells and analysis its effects on Fas-mediated programmed cell death.Results:We found an ALPS patient that harbored a heterozygous A530G mutation in the FasL gene that replaced Arg with Gly at position 156 in the protein's extracellular Fas-binding region. This produced a dominant-interfering FasL protein that bound to the wild-type FasL protein and prevented it from effectively inducing apoptosis.Conclusion:Our data explain how a naturally occurring heterozygous human FasL mutation can dominantly interfere with normal FasL apoptotic function and lead to an ALPS phenotype, designated Type Ib.

Cloning and functional identification of slc5a12 as a sodium-coupled low-affinity transporter for monocarboxylates (SMCT2)

We report in the present paper, on the isolation and functional characterization of slc5a12, the twelfth member of the SLC5 gene family, from mouse kidney. The slc5a12 cDNA codes for a protein of 619 amino acids. Heterologous expression of slc5a12 cDNA in mammalian cells induces Na+-dependent transport of lactate and nicotinate. Several other short-chain monocarboxylates compete with nicotinate for the cDNA-induced transport process. Expression of slc5a12 in Xenopus oocytes induces electrogenic and Na+-dependent transport of lactate, nicotinate, propionate and butyrate. The substrate specificity of slc5a12 is similar to that of slc5a8, an Na+-coupled transporter for monocarboxylates. However, the substrate affinities of slc5a12 were much lower than those of slc5a8. slc5a12 mRNA is expressed in kidney, small intestine and skeletal muscle. In situ hybridization with sagittal sections of mouse kidney showed predominant expression of slc5a12 in the outer cortex. This is in contrast with slc5a8, which is expressed in the cortex as well as in the medulla. The physiological function of slc5a12 in the kidney is likely to mediate the reabsorption of lactate. In the intestinal tract, slc5a12 is expressed in the proximal parts, whereas slc5a8 is expressed in the distal parts. The expression of slc5a12 in the proximal parts of the intestinal tract, where there is minimal bacterial colonization, suggests that the physiological function of slc5a12 is not to mediate the absorption of short-chain monocarboxylates derived from bacterial fermentation but rather to mediate the absorption of diet-derived short-chain monocarboxylates. Based on the functional and structural similarities between slc5a8 and slc5a12, we suggest that the two transporters be designated as SMCT1 (sodium-coupled monocarboxylate transporter 1) and SMCT2 respectively.

A Ubiquitous β-tubulin Disrupts Microtubule Assembly and Inhibits Cell Proliferation

Vertebrate tubulin is encoded by a multigene family that produces distinct gene products, or isotypes, of both the alpha- and beta-tubulin subunits. The isotype sequences are conserved across species supporting the hypothesis that different isotypes subserve different functions. To date, however, most studies have demonstrated that tubulin isotypes are freely interchangeable and coassemble into all classes of microtubules. We now report that, in contrast to other isotypes, overexpression of a mouse class V beta-tubulin cDNA in mammalian cells produces a strong, dose-dependent disruption of microtubule organization, increased microtubule fragmentation, and a concomitant reduction in cellular microtubule polymer levels. These changes also disrupt mitotic spindle assembly and block cell proliferation. Consistent with diminished microtubule assembly, there is an increased tolerance for the microtubule stabilizing drug, paclitaxel, which is able to reverse many of the effects of class V beta-tubulin overexpression. Moreover, transfected cells selected in paclitaxel exhibit increased expression of class V beta-tubulin, indicating that this isotype is responsible for the drug resistance. The results show that class V beta-tubulin is functionally distinct from other tubulin isotypes and imparts unique properties on the microtubules into which it incorporates.

Construction of multi-purpose vectors, pCNS and pCNS-D2, are suitable for collection and functional study of large-scale cDNAs

To efficiently perform collection and functional studies of large-scale cDNAs, we constructed multi-functional cDNA vectors for efficient full-length cDNA cloning, direct sequencing, easy screening, and the expression of cDNA in vitro and in vivo without subcloning the cDNA into other vectors. The constructed vectors, pCNS and pCNS-D2, contain a multi-cloning site for uni-directional full-length cDNA cloning, T7 and Sp6 RNA polymerase promoters for in vitro transcription and translation, and hCMV immediate early promoter and BGH poly(A) to allow expression in mammalian cells. Using these vectors, we constructed full-length enriched cDNA libraries containing 60–75% of the full-length cDNAs using two different oligo-capping methods. The subtracted cDNA libraries could also be constructed by removing of EF1-α cDNA, a highly expressed cDNA. In addition, we confirmed the translation of EF1-α cDNA in vitro and the expression of luciferase cDNA in mammalian cells. The expression efficiency of luciferase cDNA in different cell lines, such as HeLa, Hep3B, SNU638, and SNU668, showed that pCNS vectors can highly express target genes in different cell types. These results indicated that our multi-purpose vectors, pCNS and pCNS-D2, are useful tools for the construction of full-length cDNA libraries and high-throughput based functional study of cDNAs.

Characterization of POMT2, a novel member of the PMT protein O-mannosyltransferase family specifically localized to the acrosome of mammalian spermatids.

Over the past few years it has emerged that O-mannosyl glycans are not restricted to yeasts and fungi but are also present in higher eukaryotes, including humans. They play a substantial role in the onset of muscular dystrophy and neuronal migration disorders, like muscle-eye-brain disease. Protein O-mannosyltransferase genes (PMTs) are evolutionarily conserved from yeast to human; however, little is known about these enzymes in higher eukaryotes. In this study, we cloned the first PMT2 subfamily members from human (hPOMT2), mouse (mPomt2), and Drosophila (DmPOMT2). A detailed characterization of the mammalian POMT2, with emphasis on mouse Pomt2, shows that mammalian POMT2 is predominantly expressed in testis tissue. Due to differential transcription initiation of the mPomt2 gene, two distinct mRNA species that vary in length are formed. The shorter transcript is present in all somatic tissues examined. Expression of the corresponding hPOMT2 cDNA in mammalian cells identified POMT2 as an integral membrane protein of the endoplasmic reticulum with an apparent molecular weight of 83 kDa. The longer mPomt2 transcript is restricted to testis and encodes a testis-specific mPOMT2 protein isoform. Using in situ hybridization and immunolocalization, we demonstrate that in testis tissue mPOMT2 localizes to maturing spermatids and is abundant within the acrosome, a sperm-specific organelle essential for fertilization. Our data suggest a novel and specific role for the putative protein O-mannosyltransferase POMT2 in the maturation and/or function of sperm in mammals.

Expression and characterization of nonmammalian selenoprotein P in the zebrafish, Danio rerio.

Selenoprotein P is a protein of considerable intrigue, due to its unusual composition and requirements for its biosynthesis. Whereas most selenoproteins contain a single selenocysteine residue, the human, bovine and rodent selenoprotein P genes encode proteins containing 10-12 selenocysteines. Selenoprotein P genes have, to date, only been reported in mammals, and the function of the protein remains elusive. Herein, we report the identification and characterization of nonmammalian selenoprotein P in the zebrafish Danio rerio. Sequencing of the cDNA revealed the presence of 17 selenocysteine codons, the highest number reported in any protein. Two histidine-rich regions present in the mammalian selenoprotein P sequences are conserved in the zebrafish protein, and two SECIS elements are present in the 3' untranslated region. Whole-mount in situ hybridization of zebrafish embryos revealed high levels of expression of selenoprotein P mRNA in fertilized eggs and in the yolk sac of developing embryos. Transient transfection of the cDNA in mammalian cells resulted in efficient expression of the full-length secreted selenoprotein. A single N-glycosylation site is predicted, and shown to be utilized. Discovery of selenoprotein P in the zebrafish opens a previously unavailable avenue for genetic investigation of the functions of this unusual protein.

PB2 polymerase subunit of Thogoto virus (Orthomyxoviridae family)

Thogoto virus (THOV) represents a new genus in the family Orthomyxoviridae. The three polymerase subunits PB1, PB2, and PA initiate transcription by a unique cap-stealing mechanism that involves the cleavage of only the m(7)GpppAm structure from cellular hnRNAs. Here, we report the cloning of the longest genomic segment of THOV coding for PB2. It comprises 2,375 nucleotides encoding a single large open reading frame flanked by the conserved terminal regions typical for orthomyxoviruses. The deduced amino acid sequence corresponds to a protein of 769 amino acids, with an estimated Mr of 88,042. Expression of the PB2 cDNA in mammalian cells revealed nuclear accumulation of the recombinant protein, consistent with the replication of THOV in the cell nucleus.

Localization of the APECED protein in distinct nuclear structures.

Autoimmune-polyendocrinopathy-candidiasis-ecto-dermaldystrophy (APECED) is the only systemic autoimmune disease with a monogenic background known so far revealing no association with the major histocompatibility complex region. We have recently isolated the gene defective in this syndrome and characterized several different mutations in individuals with the disorder. The novel gene, AIRE, contains a putative bipartite nuclear targeting signal predicting a nuclear location of the corresponding protein. The presence of two PHD-type zinc finger domains as well as the newly described putative DNA-binding domain, SAND, in the amino acid sequence of the APECED protein implies that it may be involved in the regulation of gene expression. Using transient expression of AIRE cDNA in mammalian cells we demonstrate here the nuclear location of the APECED protein. Immunohistochemical staining of transfected cells revealed that most of the recombinant 58 kDa APECED protein is present in the form of nuclear dots. By double immuno-fluorescence labelling we further show that these APECED-containing structures and the previously described PML nuclear bodies are largely non-overlapping. The AIRE protein was also visualized in multiple human tissues: a subset of the cells in thymus, in spleen and in lymph node showed nuclear staining with APECED antiserum. Immunofluorescence labelling of peripheral blood mononuclear leukocytes also revealed a nuclear body-like staining pattern in a fraction of these cells. These data from both in vitro and ex vivo systems, together with the predicted structural features of the APECED protein, suggest that this protein is most probably involved in the regulation of gene expression.

Characterization of the transcription factor MTF-1 from the Japanese pufferfish (Fugu rubripes) reveals evolutionary conservation of heavy metal stress response.

The pufferfish Fugu rubripes was recently introduced as a new model organism for genomic studies, since it contains a full set of vertebrate genes but only 13% as much DNA as a mammal. Fugu genes tend to be smaller and densely spaced due to shortening of introns and intergenic spacers. We isolated the Fugu gene for the metal-responsive transcription factor MTF-1 (MTF1), a mediator of heavy metal regulation and oxidative stress response previously characterized in mammals. In addition, most of the cDNA sequence was also determined. The 780 amino acid MTF-1 protein of Fugu is very similar to that of mouse and human, with 90% amino acid identity in the DNA binding zinc finger domain and 57% overall identity. Expression of the pufferfish cDNA in mammalian cells shows that Fugu MTF-1 has the same DNA binding specificity as its mammalian counterpart and also induces transcription in response to zinc and cadmium. The protein-coding part of the Fugu MTF-1 gene spans 6.4 kb and consists of 11 exons. Upstream region and first exon constitute a CpG island. The distance between stop codon and polyadenylation motifs is >2 kb, suggesting a very long 3' untranslated mRNA region, followed by another CpG island which may represent the promoter of the next gene downstream. Part of the MTF-1 genomic structure was also determined in the mouse, and some striking similarities were found: for example, the upstream adjacent gene in both species is INPP5P, encoding a phosphatase. The mouse MTF-1 promoter is also embedded in a CpG island, which however shares no sequence similarity to the one of Fugu. The Fugu CpG island is shorter than the one of the mouse and has no elevated [G+C] content; these and other data indicate that CpG islands of fish may represent a primordial stage of CpG island evolution.

Properties of Whole Human IgG Molecules Produced by the Expression of Cloned Anti-DNA Antibody cDNA in Mammalian Cells

Antibodies to DNA are believed to play an important role in systemic lupus erythematosus (SLE). High affinity IgG antibodies which show marked specificity for double stranded DNA (dsDNA) are particularly closely linked to the occurrence and severity of tissue damage. Sequence analysis of mouse and human monoclonal antibodies has previously suggested that mutations in the complementarity determining regions (CDRs) play a major role in determining these binding properties. In many cases such mutations increase the overall number of basic residues in the CDRs. To further elucidate the role played by such mutations it is important to develop methods of expressing cloned autoantibody cDNA in the form of functional whole immunoglobulin molecules. We describe a system in which autoantibody VHand VLcDNA from monoclonal human anti-DNA antibodies, B3 and WRI176 were cloned into separate vectors which allowed their expression as whole heavy and whole light chains respectively. By cotransfecting mammalian cells with pairs of heavy and light chain vectors it was possible to produce whole IgG molecules from each of the four possible VH/VLcombinations. Only antibody produced by homologous VHand VLpairs bound DNA, suggesting that in these autoantibodies both chains are important in conferring this property.

Cloning and Structural Analysis of the cDNA and the Gene Encoding the Murine Type 1 Sigma Receptor

We have isolated a mouse cDNA which codes for a functional type 1 sigma receptor. The functional identity of the cDNA was established by expressing the cDNA in mammalian cells and measuring the cDNA-induced binding of haloperidol. Using this cDNA as the probe, we have isolated a murine genomic clone which contains the type 1 sigma receptor gene in its entirety. We have sequenced the gene completely, deduced the exon-intron organization and analyzed the promoter region sequence for transcription factor binding sites. The gene (∼7 kbp) is TATA-less but contains CCAATC and GC boxes immediately upstream of the transcription start site. The gene consists of 4 exons and 3 introns. The 5′-flanking region contains putative binding sites for AP-1, AP-2, GATA-1 and steroid receptors.

Placenta Growth Factor: Identification and Characterization of a Novel Isoform Generated by RNA Alternative Splicing

We report the isolation and characterization of a third isoform of placenta growth factor (PlGF), generated by alternative splicing of its RNA transcript. This novel form of PlGF, PlGF-3, was cloned by the polymerase chain reaction technique using a human cDNA library prepared from the terminal placental tissue. PlGF-3 contains an in-frame insertion of 72-amino acids near the C-terminal portion of PlGF-1. Southern blot analysis revealed that a single gene encoded PlGF-2 and PlGF-3. Nucleic acid sequence analysis found the insertion of 216 nucleotides of PlGF-3 between exon 4 and exon 5 of the PlGF gene. Northern blot and tissue distribution studies discovered two mRNA species for PlGF-3, which were both uniquely expressed in the placenta. Transient expression of PlGF-3 cDNA in mammalian cells showed PlGF-3 was detected in the conditioned medium as both dimers and monomers. Unlike PlGF-2, PlGF-3 lacked heparin-binding affinity. Thus, alternative splicing of PlGF RNA produces at least three polypeptides with different secretion pattern, heparin-binding affinity and dimerization properties.

A Novel Cytosolic Calcium-independent Phospholipase A2 Contains Eight Ankyrin Motifs

We report the purification, molecular cloning, and expression of a novel cytosolic calcium-independent phospholipase A2 (iPLA2) from Chinese hamster ovary cells, which lacks extended homology to other phospholipases. iPLA2 is an 85-kDa protein that exists as a multimeric complex of 270-350 kDa with a specific activity of 1 micromol/min/mg. The full-length cDNA clone encodes a 752-amino acid cytoplasmic protein with one lipase motif (GXS465XG) and eight ankyrin repeats. Expression of the cDNA in mammalian cells generates an active 85-kDa protein. Mutagenesis studies show that Ser465 and the ankyrin repeats are required for activity. We demonstrate that iPLA2 selectively hydrolyzes the sn-2 over sn-1 fatty acid by 5-fold for 1,2-dipalmitoyl phosphatidylcholine in a mixed micelle. Moreover, we found the fatty acid preference at the sn-2 position to be highly dependent upon substrate presentation. However, iPLA2 does have a marked preference for 1,2-dipalmitoyl phosphatidic acid presented in a vesicle, generating the lipid second messenger lysophosphatidic acid. Finally the enzyme is able to hydrolyze the acetyl moiety at the sn-2 position of platelet-activating factor.

Expression of Human Lysosomal Alpha-Mannosidase Activity in Transfected Murine Cells and Human Alpha-Mannosidase Deficient Fibroblasts

We studied the human lysosomal alpha-mannosidase (MANB) by expressing the putative cDNA in mammalian cells, using the eucaryotic expression vector pCDE. The construct pCDE-MANB and pSV2-Neo were cotransfected into human α-mannosidase deficient fibroblasts and into a murine cell line and selected by culture in the presence of G418. Six G418 resistant 3T3 clones had increased α-mannosidase activity 2 to 3 times above the controls. Two clones from transfected human fibroblasts showed a 2 fold increase in enzyme activity. The human MANB cDNA gene was demonstrated in the target cells by Southern blot analysis and the expression of the gene was shown by RT-PCR analysis. This study is the first to successfully express the MANB gene in a human and a murine cell line. The results confirm that the putative MANB cDNA encodes the full length of lysosomal α-mannosidase. Molecular characterization of mannosidosis and approaches to gene therapy are now possible using this cDNA.

Development of drug targeting based on recombinant expression of the chicken avidin gene.

The chemistry required for covalent biotinylation of drugs, radiopharmaceuticals and other ligands is highly developed, and a large number of biotinylated reagents can be readily synthesized. In order to investigate whether expression of avidin cDNA in mammalian cells might be useful as part of a drug targeting strategy, we transiently expressed the avidin gene in two human tumor cell lines (the cervical carcinoma cell line, HeLa, and the liver derived line, Hep G2). Avidin protein as detected by either immunohistochemistry or binding of streptavidin-biotin complexes was present and functional following transient expression. This result indicated that the mechanisms underlying avidin oligomerization which are necessary for proper protein folding are present within mammalian carcinoma cell lines. Next, we generated a producer cell line (derived from psi2) capable of releasing a recombinant retrovirus encoding chicken avidin, and a tumorigenic murine breast cancer cell line (16/C) with stable avidin expression. We show that these cell lines are suitable for conferring functional expression of avidin in vitro. These experiments establish a means by which avidin gene expression can be explored as a mechanism for targeted gene delivery of biotin-derivitized drugs in vitro, and have important implications for utilization of this strategy in vivo.

The transcription factor E2F is required for S phase during Drosophila embryogenesis.

Overexpression of the E2F-1 cDNA in mammalian cells disrupts normal control of the cell cycle and drives cells into S phase. Whereas eliminating E2F activity would test its inferred involvement in the G1-S transition, elimination is complicated by the existence of gene families encoding mammalian E2F. Here we identify mutations in a single essential Drosophila gene, dE2F, that encodes a homolog of the mammalian E2F gene family. Embryos homozygous for null mutations of dE2F complete early cell cycles, presumably using maternal contributions of gene products, but DNA synthesis falls to virtually undetectable levels in cycle 17. Mutant embryos also lack the pulses of coordinate transcription of genes encoding replication functions that usually accompany each transition from quiescence to S phase. We conclude that in most cells dE2F is essential for a G1-S transcriptional program and for G1-S progression.