Human Y1 Research Articles

Expression Cloning and Pharmacological Characterization of a Human Hippocampal Neuropeptide Y/Peptide YY Y2 Receptor Subtype

The pancreatic polypeptide family includes neuropeptide Y (NPY), one of the most abundant neuropeptides in the mammalian nervous system, as well as peptide YY (PYY) and pancreatic polypeptide (PP). This peptide family is involved in numerous physiological processes such as memory, pain, blood pressure, appetite, anxiety, and circadian rhythm. Of the multiple Y-type receptors proposed for PP family members, only the Y1 subtype was cloned previously. We now report the isolation of a human Y2 (hhY2) receptor cDNA by expression cloning from a human hippocampal cDNA library, using a 125I-PYY binding assay. hhY2 cDNA encodes a predicted protein of 381 amino acids with low amino acid identity to the human Y1 receptor (31% overall; 41% transmembrane). 125I-PYY binding to transiently expressed hY2 receptors was saturable (pKd = 10.17) and displaceable by human PP family members in rank order: PYY (pKi = 9.47) approximately NPY (pKi = 9.27) >> PP (pKi < 6) and by peptide analogs: NPY2-36 (pKi = 8.80) approximately NPY13-36 (pKi = 8.55) approximately C2-NPY (pKi = 8.54) > NPY26-36 (pKi = 6.51) approximately [Leu31,Pro34]NPY (pKi = 6.23). Human PYY decreased [cAMP] and increased intracellular [Ca2+] in hY2-transfected 293 cells.

Characterization of neuropeptide Y (NPY) receptors in human cerebral arteries with selective agonists and the new Y1 antagonist BIBP 3226

1. We have characterized pharmacologically the receptor subtype(s) responsible for the neuropeptide Y (NPY)-induced vasoconstriction in human cerebral arteries. NPY, PYY and several of their derivatives with well defined affinities at the known Y1 and Y2 receptor subtypes were used. Moreover, we tested the ability of the new Y1 receptor antagonist, BIBP 3226, to antagonize the NPY-induced cerebral vasoconstriction. 2. NPY, PYY and their agonists with high affinities at the Y1 receptor subtype ([Leu31-Pro34]-NPY and [Leu31-Pro34]-PYY) elicited strong, long lasting and concentration-dependent contractions of human cerebral arteries. Compounds with Y2 affinity such as PYY3-36 or NPY13-36 either elicited a submaximal contraction at high concentrations or failed to induce any significant vasomotor response. Also, the application of NPY or the specific Y1 agonist, [Leu31-Pro34]-NPY, to human cerebral vessels pretreated with the Y1 agonist, NPY13-36, resulted in contractile responses identical to those obtained when these compounds were tested without prior application of NPY13-36. 3. The order of agonist potency at the human cerebrovascular receptor was: [Leu31-Pro34]-NPY = [Leu31-Pro34]-PYY > or = NPY > PYY > PYY3-36 > > > NPY13-36, which corresponded to that reported previously at the neuronal and vascular Y1 receptors. 4. Increasing concentrations (10(-9)-10(-6) M) of the Y1 receptor antagonist, BIBP 3226, to human cerebral vessels caused a parallel and rightward shift in the NPY dose-response curves without any significant change in the maximal contractile response. The calculated pA2 was 8.52 +/- 0.13, a value compatible with the reported affinity at the rodent and human Y1 receptor. 5. We conclude that Y1 receptors exclusively, mediate the NPY-induced contraction in human cerebral arteries and we show that BIBP 3226 is a potent and competitive antagonist of this YI-mediated vasoconstriction.

Cloning and Functional Expression of a cDNA Encoding a Human Type 2 Neuropeptide Y Receptor

Neuropeptide Y (NPY) is a 36-amino acid polypeptide that is widely distributed in the central nervous system and periphery. Pharmacological studies have suggested that there are at least three receptor subtypes, Y1, Y2, and Y3. Cloning of the Y1 subtype has been reported previously. Here we report the isolation by expression cloning of a cDNA encoding a human NPY receptor displaying a pharmacology typical of a Y2 receptor. COS-7 cells transfected with the cDNA express high affinity binding sites for NPY, peptide YY, and NPY13-36, whereas [Leu31,Pro34]NPY binds with lower affinity. The receptor is 381 amino acids in length and has seven putative transmembrane regions typical of G-protein-coupled receptors. Comparison of the amino acid sequence of this Y2 receptor to that of the human Y1 receptor indicates that the two receptors are 31% identical at the amino acid level. Northern blot analyses reveal a single 4-kilobase mRNA species and indicate that the messenger RNA is present in many areas of the central nervous system. NPY induced calcium mobilization and inhibited forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells that stably express the Y2 receptor cDNA, indicating that the recombinant Y2 receptor is functionally coupled to second messenger systems.

Role of a hydrophobic pocket of the human Y1 neuropeptide Y receptor in ligand binding

We are investigating the nature of the chemical interactions between the neuropeptide Y (NPY) and its cell surface receptor (Y1). A previous study involving site-directed mutagenesis and computer-aided modelling (Walker et al., 1994) suggested that the C-terminal Tyr36 of NPY, known to be a key residue for receptor binding, might dock at a pocket formed by hydrophobic amino acids of transmembrane domains (TM) 1, 2, 6 and 7 of the Y1 receptor. To investigate which residues were required for ligand binding, we mutated the sequences encoding F41, L43, F96, Y100, F286 and H298 of the human Y1 receptor. The mutant cDNAs were transiently expressed in Hela cells and the ability of the encoded proteins to bind NPY was evaluated. Replacing F41, L43 or F96 with alanines had no effect on NPY binding. On the contrary, Y100, F286 and H298 appeared to be residues critical for ligand binding. In particular, the removal of the hydroxyl group of Y100 (Tyr100 → Phe100 mutation) yielded a protein devoid of affinity for the ligand. The level of expression and the presence on the cell surface of mutants lacking NPY binding activity was assessed by immunological techniques. In addition, we tested the ability of synthetic analogues of neuropeptide Y with substitutions at position 36 to bind to the Y1 receptor. To get spatial insight into the relative positions of the above mentioned residues we constructed a molecular model of the interaction between NPY:Y36 and the elements of the hydrophobic pocket surrounding this residue.

A perinucleolar compartment contains several RNA polymerase III transcripts as well as the polypyrimidine tract-binding protein, hnRNP I.

We have investigated the subcellular organization of the four human Y RNAs. These RNAs, which are transcribed by RNA polymerase III, are usually found complexed with the Ro autoantigen, a 60-kD protein. We designed 2'-OMe oligoribonucleotides that were complementary to accessible single-stranded regions of Y RNAs within Ro RNPs and used them in fluorescence in situ hybridization. Although all four Y RNAs were primarily cytoplasmic, oligonucleotides directed against three of the RNAs hybridized to discrete structures near the nucleolar rim. We have termed these structures "perinucleolar compartments" (PNCs). Double labeling experiments with appropriate antisera revealed that PNCs are distinct from coiled bodies and fibrillar centers. Co-hybridization with a genomic DNA clone spanning the human Y1 and Y3 genes showed that PNCs are not stably associated with the transcription site for these Y RNAs. Although 5S rDNA was often located near the nucleolar periphery, PNCs are not associated with 5S gene loci. Two additional pol III transcripts, the RNA components of RNase P and RNase MRP, did colocalize within PNCs. Most interestingly, the polypyrimidine tract-binding protein hnRNP I/PTB was also concentrated in this compartment. Possible roles for this novel nuclear subdomain in macromolecular assembly and/or nucleocytoplasmic shuttling of these five pol III transcripts, along with hnRNP I/PTB, are discussed.

Coupling of the human Y2 receptor for neuropeptide Y and peptide YY to guanine nucleotide inhibitory proteins in permeabilized SMS-KAN cells.

Using guanine nucleotides, pertussis toxin, and specific antisera against the COOH-terminals of the alpha-subunits of Gi1/2, Gi3, and G(o), the binding and biological response of the Y2 receptor (Y2R) for peptide YY (PYY) was probed in SMS-KAN neuroblastoma cells. The specific binding of radiolabeled PYY exhibited a single apparent dissociation constant, KD = 76 pM for intact cells and KD = 906 pM for permeabilized cells. However, other data suggested existence of multiple receptor affinity states. A shift in KD and a decrease in apparent number of binding sites (Bmax) was observed in permeabilized cells when incubated with guanine nucleotides. By contrast, in membrane preparations guanine nucleotides induced only a decrease in Bmax. In intact cells, agonist exposure inhibited the intracellular accumulation of forskolin-stimulated cyclic AMP by 80% (IC50 = 420 nM) compared with 94% inhibition (IC50 = 380 nM) in permeabilized cells. In permeabilized cells, preincubation with antisera against alpha i1/2 and alpha i3 blocked the functional response of PYY, with anti-alpha i3 being the most potent; whereas anti-alpha o failed to affect the cyclic AMP levels. These results suggest that permeabilized SMS-KAN cells serve as a good model system for analysis of Y2R binding kinetics and functional response and that the Y2R interacts directly with several different GiS (but not G(o)).

Characterization of the human Y1 neuropeptide Y receptor expressed in insect cells

We have expressed the human Y1 NPY receptor in insect cells using a recombinant baculovirus (BacY1). Non-linear curve fitting of competition binding data indicates the presence of 500 000–750 000 saturable NPY binding sites per cell. The affinity of the recombinant Y1 receptor for NPY ( K d = 0.38 ± 0.8 nM) was identical to the natural receptor. We used a foreign epitope to characterize, immunopurify; and localize the recombinant protein. Cross-linking experiments identified a 65 kDa band as the major NPY binding species. Confocal microscopy indicated that although some recombinant proteins are detectable as early as 12 h post-infection, significant expression at the cell surface is only seen 24–48 h post-infection. We also describe a procedure to treat infected Sf21 cells in such a way that they can be frozen and stored at −80°C for many months before being used for binding studies.

Receptor binding profiles of NPY analogues and fragments in different tissues and cell lines.

To investigate receptor selectivity and possible species selectivity of a number of NPY analogues and fragments, receptor binding studies were performed using cell lines and membranes of several species. NPY displays 4-25-fold higher affinity for the Y2 receptor than for the Y1 receptor. The affinity of [Leu31,Pro34]NPY is 7-60-fold higher for the Y1 receptor when compared with the Y2 subtype. Species selectivity within the Y2 receptors is demonstrated by PYY(3-36), NPY(2-36), NPY(22-36), and NPY(26-36). It is shown that NPY(22-36) is species selective for the human Y2 subtype (K1 of 0.3 nM) compared with the rabbit and rat Y2 receptor (K1 of 2 and 10 nM, respectively). PYY(3-36) displays highest affinity for the human and rabbit Y2 subtype (K1 of 0.03 and 0.17 nM). The screening of NPY analogues and fragments revealed that highest affinity for the human Y2 receptor is shown by NPY(2-36) and PYY(3-36). In addition, PYY(3-36) and NPY(2-36) are not only subtype selective, but also species selective.

Human Neuropeptide Y1 Receptor Expressed inEscherichia coliRetains Its Pharmacological Properties

The coding region of the gene for the human neuropeptide Y (NPY) Y1 receptor was fused to the maltose binding protein gene. Expression of this transcription unit, after derepression with isopropyl beta-D-thiogalactopyranoside, was evidenced by the appearance of a protein of higher molecular weight, as well as the native maltose binding protein, both of which were immunoreactive with anti-maltose binding protein antibodies. Specific [125I]NPY binding activity was found associated mainly with the inner bacterial membrane fraction, suggesting that the receptor is correctly folded in this membrane. Competition binding experiments using NPY Y1- and Y2-specific ligands clearly exhibited a NPY Y1 specific pharmacological profile with Kd values indistinguishable from those of the native receptor. These results suggest that the membrane environment required by the human NPY Y1 receptor for specific high-affinity ligand binding is conserved in this heterologous bacterial system and can be used for detailed analyses of ligand-receptor interaction and drug screening.

Complete L‐Alanine Scan of Neuropeptide Y Reveals Ligands Binding to Y1 and Y2 Receptors with Distinguished Conformations

The synthesis of more than fifty 36-residue oligopeptide analogs of neuropeptide Y (NPY) and their affinity to human Y1 and Y2 receptors is described. Each amino acid of the natural sequence was replaced by L-alanine, the four alanine residues at position 12, 14, 18 and 23 were replaced by glycine. Additional residues were exchanged to closely related ones in order to characterize the prerequisites for binding. A combination of automated single and multiple peptide synthesis using fluoren-9-ylmethoxycarbonyl/tert-butoxy strategy was applied. The purified peptides were characterized by electrospray mass spectrometry, analytical HPLC and amino acid analysis. Binding was investigated by displacement of 125I-labelled neuropeptide Y from human neuroblastoma cell lines SK-N-MC and SMS-KAN. Whereas Pro2 and the integrity of the neuropeptide Y loop is important for the binding to the Y1 receptor, exchanges within the C-terminal helix affect the affinity to the Y2 receptor. The C-terminal pentapeptide amide is important for both receptors and probably represents the binding site. However, Arg33 and Arg35 may not be exchanged by L-alanine in the Y1 system, whereas Arg35 and Tyr36 are the most susceptible residues in the Y2 system. In order to distinguish between conformational effects and direct hormone/receptor interaction via the side chains of neuropeptide Y, circular dichroic studies of the alanine-containing peptides were performed and structure affinity relationships are discussed. Comparing the affinities of the neuropeptide Y analogs to Y1 and Y2 receptors significant differences were found for the two binding sites, which suggests a different active conformation of neuropeptide Y at the two subtypes of receptors. Using molecular dynamics calculations, two distinct conformations were identified which are in good agreement with the data obtained by structure/affinity investigations.

Characterization of a Y1-preferring NPY/PYY receptor in HT-29 cells.

Equilibrium binding studies showed that butyrate-treated HT-29 cells express a high-affinity 125I-labeled peptide YY (125I-PYY) binding site with a dissociation constant of 0.32 +/- 0.12 nM (mean +/- SE, n = 4). This site was Y1 preferring because neuropeptide Y (NPY) and the Y1-selective agonist [Leu31,Pro34]NPY were equipotent to PYY at displacing 125I-PYY; PYY-(13-36) and pancreatic polypeptide were > 1,000- and 10,000-fold less potent at displacing the radioligand. PYY and [Leu31,Pro34]NPY inhibited forskolin-stimulated adenosine 3',5'-cyclic monophosphate production 63% and 48%, respectively, with a half-maximal inhibitory concentration between 0.1 and 1.0 nM. PYY and [Leu31,Pro34]NPY had no effect on release of intracellular calcium alone or on the increase in intracellular calcium concentration caused by carbachol or neurotensin. Northern blot analysis of poly(A)+ RNA from HT-29 cells demonstrated a single transcript of 2.5 kb that hybridized to a human Y1-receptor cDNA probe. Sequence analysis of a reverse transcription-polymerase chain reaction product amplified with primers based on human Y1-receptor cDNA confirmed that these cells contained mRNA encoding the human Y1 receptor. These studies show that butyrate-treated HT-29 cells constitutively express the Y1-preferring NPY/PYY receptor and Y1 mRNA and provide a new model for studies of PYY-regulated epithelial cell function and tissue-specific expression of the human Y1-receptor gene.

Analysis of the intracellular localization and assembly of Ro ribonucleoprotein particles by microinjection into Xenopus laevis oocytes.

Xenopus laevis oocytes have been used to determine the intracellular localization of components of Ro ribonucleoprotein particles (Ro RNPs) and to study the assembly of these RNA-protein complexes. Microinjection of the protein components of human Ro RNPs, i.e., La, Ro60, and Ro52, in X. laevis oocytes showed that all three proteins are able to enter the nucleus, albeit with different efficiencies. In contrast, the RNA components of human Ro RNPs (the Y RNAs) accumulate in the X. laevis cytoplasm upon injection. Localization studies performed at low temperatures indicated that both nuclear import of Ro RNP proteins and nuclear export of Y RNAs are mediated by active transport mechanisms. Immunoprecipitation experiments using monospecific anti-La and anti-Ro60 antibodies showed that the X. laevis La and Ro60 homologues were cross-reactive with the respective antibodies and that both X. laevis proteins were able to interact with human Y1 RNA. Further analyses indicated that: (a) association of X. laevis La and Ro60 with Y RNAs most likely takes place in the nucleus; (b) once formed, Ro RNPs are rapidly exported out of the nucleus; and (c) the association with La is lost during or shortly after nuclear export.

The human Y4 small cytoplasmic RNA gene is controlled by upstream elements and resides on chromosome 7 with all other hY scRNA genes.

Ro ribonucleoproteins (RNP) constitute a class of evolutionarily conserved small cytoplasmic (sc) RNPs whose functions are unknown. In human cells four distinctive scRNAs designated hY1, hY3, hY4 and hY5 are synthesized by RNA polymerase III (pol III) and accumulate as components of Ro scRNPs. The previously isolated hY1 and hY3 genes contain upstream sequences similar to the class III promoters for U6 and 7SK snRNAs. Additional mammalian Y scRNA genes have been refractory to cloning due to interference from numerous hY-homologous pseudogenes and studies of hY RNA genes have been sparse. Although homologs of hY1 and hY3 RNAs exist in rodent cells, the smaller Y4 and Y5 RNAs do not which has allowed us to localize the hY4 scRNA gene to human chromosome 7 by assaying for its transcript in rodent X human somatic cell hybrids (SCH). A chromosome 7-enriched yeast artificial chromosome (YAC) library was then screened and the authentic hY4 sequence was isolated by strepavidin--biotin-mediated hybrid-selection followed by poly(dA)-tailing and hemispecific PCR. The region upstream of the hY4 sequence contains a TATAAAA motif centered at -26, a candidate proximal sequence element at -63, and three octamer-like sequences located between -260 and -200. hY4 RNA is readily detectable on Northern blots after transient transfection of the hY4 gene into mouse cells but not after transfection of a construct in which the 5' flanking region was deleted. SCHs and chromosome 7-enriched YACs were used to demonstrate that all four hY RNA genes reside on human chromosome 7.

Acidic residues in extracellular loops of the human Y1 neuropeptide Y receptor are essential for ligand binding.

To investigate whether negatively charged residues of the human Y1 neuropeptide Y (NPY) receptor are required for ligand binding, a series of mutants were constructed in which aspartic acid and glutamic acid residues present in putative extracellular domains of the Y1 receptor were systematically replaced by alanines. The mutant cDNAs were transiently expressed in HeLa cells using a vaccinia virus-derived expression system, and their ability to bind NPY was evaluated. The level of expression of mutants unable to bind NPY was also tested immunologically. In addition, the ability of the mutant proteins to be recruited to the cell surface was assessed by confocal microscopy. Substitution of aspartic acids and glutamic acids of the N-terminal first extracellular domain had no effect on binding. On the other hand, substitution of acidic residues present in the second, third, and fourth extracellular loops resulted in proteins unable to bind 125I-NPY. These results demonstrate that the extracellular loops of the human Y1 NPY receptor are essential portions of its ligand binding domain.

Molecular cloning, characterization, and localization of the human homolog to the reported bovine NPY Y3 receptor: lack of NPY binding and activation.

A cDNA clone encoding the human homolog of the bovine cDNA clone LCR1 was isolated from a human lung cDNA library. The 1,670-bp-long nucleotide sequence predicts a single open reading frame of 352 amino acids, with a 92% amino acid identity to a bovine sequence reported to represent the neuropeptide Y (NPY) Y3 receptor. The amino acid sequence shares features common to many other G-protein-coupled receptors, including the seven transmembrane regions and putative glycosylation and phosphorylation sites. Polymerase chain reaction (PCR) analysis of human-hamster hybrid cell DNA reveals that the corresponding gene is located on human chromosome 2. Although the ligand for the bovine receptor has previously been identified as NPY in binding studies, extensive analysis with the human homolog transfected in several different cell lines failed to confirm this classification. Furthermore, the receptor shows 36% identity to both the human interleukin-8 (IL-8) and angiotensin II receptors but only 21% identity to the human NPY Y1 receptor. In addition, NPY and a number of other ligands fail to induce any change in cytosolic calcium levels in transfected cells, suggesting that this clone represents a novel neuropeptide receptor.

Expression of the human neuropeptide tyrosine Y1 receptor.

Neuropeptide tyrosine (NPY) is the predominant peptide in the innervation of many human tissues and is considered to play a role in the regulation of blood flow, gastrointestinal secretion and motility, and renal function. Three NPY receptors have been identified (Y1, Y2, and Y3) and the cDNAs encoding the human Y1 and bovine Y3 receptors have recently been cloned. We have demonstrated the expression of the Y1 receptor subtype in several fetal and adult human tissues, including the colon, kidney, adrenal gland, heart, and placenta. A single transcript was identified (approximately 2.2 kb) and localized in tissue sections by in situ hybridization. In the colon the receptor is expressed in the mucosa and basal glands, as well as the myenteric and submucous plexuses. Y1 receptor mRNA was detected in renal collecting ducts, loop of Henle, and juxtaglomerular apparatus and in the syncytiotrophoblast layer of placental villi. Fetal aorta and adult intramyocardial, colonic, and renal blood vessels also exhibited receptor expression, localized to the intima as well as the media. The distribution of Y1 receptor expression correlates with that of NPY-immunoreactive nerves and the apparent actions of NPY in the intestine, kidney, and heart. Although the placenta is devoid of nerves, an NPY-like transcript was detected in the villous trophoblast layer. The results indicate a tissue-specific regulation of NPY Y1 receptor expression.

Glycosylation of annexin I and annexin II

Human placental annexin I and annexin II were shown to be glycosylated by one-dimensional affinity blotting with the lectin concanavalin A, which recognizes D-mannose and D-glucose residues. Further evidence that annexin I and annexin II are glycosylated was provided by the finding that these proteins incorporated D-[2,6- 3H]mannose and D-[6- 3H]glucose when they were biosynthesized by the human squamous carcinoma cell line SqCC Y1 . Annexin I and annexin II could be rapidly purified from a human placental membrane extract by concanavalin A-Sepharose, which indicated that these proteins contain two biantennary mannosyl residues.

Cloning and functional expression of a human neuropeptide Y/peptide YY receptor of the Y1 type.

Neuropeptide Y (NPY) and peptide YY (PYY) are structurally related peptides that primarily function as neurotransmitter and gastrointestinal hormone, respectively. Previous functional and binding data have indicated the existence of at least three distinct receptor types, Y1, Y2, and Y3, for NPY and/or PYY in mammals. We describe here a human Y1 cDNA clone, hY1-5, isolated from a fetal brain library. The human Y1 receptor consists of 384 amino acids and has seven putative transmembrane domains like other members of the G-protein-coupled superfamily of receptors. In the region spanning the transmembrane domains, the Y1 receptor displays 29% sequence identity to human tachykinin receptors, but it only shows 21% and 23% homology with proposed bovine (LCR1) and Drosophila (PR4) NPY receptor clones, respectively. Northern blot analysis of a human neuroblastoma cell line, SK-N-MC, previously used by many investigators as a model system for studies on the Y1 receptor, revealed a single 3.5-kilobase mRNA species. Reverse transcriptase-polymerase chain reaction analysis indicated expression also in human cultured vascular smooth muscle cells, supporting the view that the Y1 receptor is associated with NPY/PYY-evoked vasoconstriction. When expressed in COS1 cells, hY1-5 conferred specific 125I-PYY binding sites with displacement patterns characteristic of the Y1 receptor, i.e. PYY greater than or equal to NPY greater than or equal to [Leu31,Pro34]NPY much greater than NPY2-36 greater than C2NPY greater than pancreatic polypeptide greater than NPY13-36 greater than NPY18-36. Moreover, in the Y1 receptor-transfected COS1 cells, but not in type 1 angiotensin II receptor-transfected control cells, NPY and PYY accelerated 45Ca2+ influx and inhibited forskolin-stimulated cAMP accumulation, both phenomena being characteristic of the mammalian Y1 receptor.

Molecular analysis of the 60-kDa human Ro ribonucleoprotein.

Ro, or Sjogren syndrome type A (SS-A), antigen is the most prevalent of the human systemic autoimmune specificities and exists as an inabundant ribonucleoprotein complex (RNP) composed of a 60,649-Da protein, as defined here by cDNA cloning, and the human Y RNAs. The recombinant 60-kDa Ro protein and human Y1 RNA were reconstituted in vitro, and the binding was enhanced by divalent cations. A region of the Ro amino acid sequence revealed a resemblance to the RNP consensus motif found in most RNA-binding proteins. In addition, Ro contained a potential "zinc-binding finger" motif that was distinct from the RNP consensus region and that may participate in the interaction with human Y RNAs or with other proteins. The recombinant Ro fusion protein also proved useful for the detection of autoantibodies in the sera of patients with autoimmune disorders. Possible functions of the Ro RNPs and their relationship to RNA polymerase III transcription are discussed.

Introduction of a Cloned Gene into Fertilized Mouse Eggs and its Expression*

Abstract In order to analyze the role of enhancer sequence for the tissue‐specific and stage specific expression of immunoglobulin gene in normal diploid cells we have introduced cloned human y1 immunoglobulin genes into fertilized mouse eggs. Human y1 genes lacking enhancer sequence were not expressed in any tissues of transgenic mice. On the other hand human y1 genes containing enhancer sequence were expressed only in B lymphocytes. Levels of HIGI mRNA and percentage of human y chain producing cells in spleen cells were increased up to 50 times after treatment with lipopolysaccharide but not with Concanavalin A, suggesting B cell specific and regulated expression of HIGI gene. Human y chain producing cells were only found in the periphery of germinal center of the white pulp in histological section of the spleen suggesting stage‐specific expression. Human y chains appeared to be coupled with mouse light chains to form a complete IgG molecule and are secreted into cell supernatant. The production and secretion of endogenous immunoglobulin heavy and light chains in transgenic mice appeared to be the same as in normal mice. About one‐seventh of spleen cells that are producing endogenous mouse heavy chain produced human y1 chain at the same time. But no cell which produce only human y chain was observed. These results suggest that human γ chain expression had no effect on mouse immunoglobulin expression.