High Degree Of Sequence Identity Research Articles

The Chick Brachyury Gene: Developmental Expression Pattern and Response to Axial Induction by Localized Activin

The mouse Brachyury gene (T) is required in notochord differentiation and posterior mesoderm formation during axial development. We have isolated the chick homologue of T(Ch-T) and determined its putative protein sequence and expression pattern during embryogenesis. Ch-T is expressed in the epiblast close to and within the primitive streak, in early migrating mesoderm and in the notochord. In later stages Ch-T expression is found in the tall bud and in the entire notochord. The notochord expression ceases in an anterior-posterior wave when the formation of the body anlage is completed. This pattern is consistent with those reported for the expression of the mouse T gene and the T homologues of Xenopus laevis and zebrafish, suggesting that the mechanisms of embryonic pattern formation are highly conserved in all vertebrates. The N-terminal half of Ch-T shows a very high degree of sequence identity with the corresponding region of mouse T which has DNA-binding activity, and with the N-terminal half of Xenopus (Xbra) and zebrafish (Ntl) T protein. Finally, we have analyzed the effects of activin A on Ch-T induction and axis formation. Localized activin A treatment of prestreak blastoderms results in ectopic Ch-T expression that correlates with formation of second primitive streaks or with repositioning of the site of single streak origin (Cooke et al., 1994). These results strengthen the previous evidence that Brachyury activation is an early response to axis-inducing signals in vivo.

Molecular Cloning and Characterization of the G Protein γ Subunit of Cone Photoreceptors

The phototransduction process in cones has been proposed to involve a G protein that couples the signal from light-activated visual pigment to the effector cyclic GMP phosphodiesterase. Previously, we have identified and purified a G beta gamma complex composed of a G beta 3 isoform and an immunochemically distinct G gamma subunit (G gamma 8) from bovine retinal cones (Fung, B. K.-K., Lieberman, B. S., and Lee, R. H. (1992) J. Biol. Chem. 267, 24782-24788; Lee, R. H., Lieberman, B.S., Yamane, H. K., Bok, D., and Fung, B. K.-K. (1992a) J. Biol. Chem. 267, 24776-24781). Based on the partial amino acid sequence of this cone G gamma 8, we screened a bovine retinal cDNA library and isolated a cDNA clone encoding G gamma 8. The cDNA insert of this clone includes an open reading frame of 207 bases encoding a 69-amino acid protein. The predicted protein sequence of G gamma 8 shares a high degree of sequence identity (68%) with the G gamma (G gamma 1) subunit of rod transducin. Similar to rod G gamma 1, it terminates in a CIIS motif that is the site for post-translational modification by farnesylation. Messenger RNA for G gamma 8 is present at a high level in the retina and at a very low level in the lung, but is undetectable in other tissues. Immunostaining of bovine retinal sections with an antipeptide antibody against the N-terminal region of G gamma 8 further shows a differential localization of G gamma 8 to cones with a pattern indistinguishable from that of G beta 3. This finding suggests that G beta 3 gamma 8 is a component of cone transducin involved in cone phototransduction and color vision.

New mobile gene cassettes containing an aminoglycoside resistance gene, aacA7, and a chloramphenicol resistance gene, catB3, in an integron in pBWH301

The multidrug resistance plasmid pBWH301 was shown to contain a sull-associated integron with five inserted gene cassettes, aacA7-catB3-aadB-oxa2-orfD, all of which can be mobilized by the integron-encoded DNA integrase. The aadB, oxa2, and orfD cassettes are identical to known cassettes. The aacA7 gene encodes a protein that is a member of one of the three known families of aminoglycoside acetyltransferases classified as AAC(6')-I. The chloramphenicol acetyltransferase encoded by the catB3 gene is closely related to members of a recently identified family of chloramphenicol acetyltransferases. The catB3 gene displays a relatively high degree of sequence identity to a chromosomally located open reading frame in Pseudomonas aeruginosa, and this may represent evidence for the acquisition by a cassette of a chromosomal gene.

Structure and expression of chicken protein kinase PITSLRE-encoding genes

The human PITSLRE protein kinases (PK), members of the p34 cdc2 kinase family named according to the single amino acid (aa) code of an important (PSTAIRE) regulatory region [Meyerson et al., EMBO J. 11 (1992) 2909–2917], are candidate tumor suppressor gene(s) localized to human chromosome 1p36.2 and a syntenic region of mouse chromosome 4 [Lahti et al., Nature Genet. 7 (1994) 370–375; Mock et al., Mammal. Genome 5 (1994) 191–192]. At least ten isoforms of this PK family are expressed from three duplicated and tandemly linked genes in humans [Xiang et al., J. Biol. Chem. 269 (1994) 15786–15794]. We have now isolated two different species of PITSLRE PK cDNAs from chicken that encode identical polypeptides, but are clearly expressed from different genes, based on nucleotide (nt) differences. Isolation of one of the corresponding chicken PITSLRE PK genes confirms that only one of the two species of PITSLRE mRNA is expressed from this gene. Comparison of the predicted avian PITSLRE PK as sequence to human and mouse sequences shows a high degree of sequence identity (>91%). Like humans, the PITSLRE PK genes in chickens must be closely linked, based on fluorescent in situ hybridization (FISH) localization of these genes to a single chicken microchromosome. PITSLRE PK mRNAs are expressed in two avian B- and T-cell lines. These results suggest that the PITSLRE PK gene family has been well conserved evolutionarily, that the gene duplication observed in humans is not a recent event, and that expression of redundant PITSLRE mRNAs is observed in different vertebrate species.

Cloning and Expression of Hydrolase C, a Member of the Rat Carboxylesterase Family

Using polymerase chain reaction (PCR), we have isolated a cDNA that encodes a rat liver carboxylesterase. This novel enzyme, designated hydrolase C, is structurally very similar to hydrolase B, a microsomal carboxylesterase expressed in rat liver and kidney. Hydrolase B and C are 96% identical in nucleotide sequence and 93% identical in deduced amino acid sequence. Both enzymes have an 18-amino-acid signal peptide at the N-terminus. The C-terminus of hydrolase B and C contains an HXEL consensus sequence for retaining proteins in the endoplasmic reticulum. As expected, when the cDNA encoding hydrolase C was expressed in a baculovirus/Sf21 cell system, the recombinant enzyme was localized in the endoplasmic reticulum. Hydrolase B and C both have putative N-linked glycosylation sites at Asn 1 and Asn 61. The active site of hydrolase B and C appears to be composed of a nucleophile, Ser 203, a basic residue, His 448, and an acidic residue, either Asp 97 or Glu 228. Based on cloning experiments, restriction endonuclease mapping and Northern blotting, hydrolase B is expressed in both rat liver and kidney, whereas hydrolase C is expressed predominantly, perhaps exclusively, in liver. When expressed in Escherichia coli, hydrolase C was catalytically inactive and unstable, but when expressed in the baculovirus/Sf21 cell system hydrolase C it was stable and catalytically active toward 1-naphthylacetate and esters of para-nitrophenol. Hydrolase C is the fourth member of the rat carboxylesterase family to be cloned and sequenced. In terms of nucleotide and deduced amino acid sequence, hydrolase C is highly similar to hydrolase B, but differs from hydrolase B in terms of its catalytic activity and tissue distribution. Recombinant hydrolase C has properties similar to those described for esterase RL2, which was purified from rat liver microsomes by Hosokawa et al. ( Arch. Biochem. Biophys. 277, 219-227, 1990), although additional studies will be required to establish conclusively the identity of this enzyme. The high degree of sequence identity (96%) between hydrolase B and C, particularly in the 3′ untranslated region, suggests that the genes encoding these two carboxylesterases evolved by duplication and divergence of a common ancestral gene.

Characterisation of the telomeres at opposite ends of a 3 Mb Theileria parva chromosome.

Bacteriophage lambda clones containing Theileria parva genomic DNA derived from two different telomeres were isolated and the nucleotide sequences of the telomeric repeats and adjacent telomere-associated (TAS) DNA were determined. The T.parva telomeric repeat sequences, a tandem array of TTTTAGGG or TTTAGGG interspersed with a few variant copies, showed a high degree of sequence identity to those of the photosynthetic algae Chlamydomonas reinhardtii (97% identity) and Chlorella vulgaris (87.7% identity) and the angiosperm Arabidopsis thaliana (84.4% identity). Unlike most organisms which have been studied, no significant repetitive sequences were found in the nucleotide sequences of TAS DNA located centromere-proximal to the telomeric repeats. Restriction mapping and hybridisation analysis of lambda EMBL3 clones containing 16 kilobases of TAS DNA derived from one telomere suggested that they did not contain long regions of repetitive DNA. The cloned TAS DNAs were mapped to T.parva Muguga genomic SfiI fragments 8 and 20, which are located at opposite ends of the largest T.parva chromosome. A 126 bp sequence located directly centromere-proximal to the telomeric repeats was 94% identical between the two cloned telomeres. The conserved 126 bp sequence was present on all T.parva Muguga telomeric SfiI fragments.

Localization of the Tp53 gene on Syrian hamster chromosome 9 by fluorescence in situ hybridization.

Mutations of the Tp53 tumor suppressor gene have been detected in 50% of all human cancers. The normal Tp53 gene product acts as a negative regulator of cell division or is associated with abnormal growth in certain differentiated cell types. The Tp53 gene was mapped by FISH to Syrian hamster chromosome 9 bands qb2.2-->2.3 using a genomic DNA probe with a high degree of sequence identity to the human Tp53 gene.

Hedgehog Signalling in Drosophila and Vertebrate Development

The hedfielwg (hh) gene family encode secreted proteins that are involved in a number of developmental processes in vertebrates and invertebrates. These include the patterning of the vertebrate neural tube, somites, limb and brain and the Drosophila embryonic epidermis and larval imaginal discs. Hh proteins show a high degree of sequence identity within and between species. Recent analysis has demonstrated that this family of molecules may act in a mechanistically similar way to provide positional signalling within different phyla.

Contribution of cadherins to directional cell migration and histogenesis in Xenopus embryos.

Perturbation of adhesion mediated by cadherins was achieved by over-expressing truncated forms of E- and EP-cadherins (in which the extracellular domain was deleted) in different blastomeres of stage 6 Xenopus laevis embryos. Injections of mRNA encoding truncated E- and EP-cadherins into A1A2 blastomeres resulted in inhibition of cell adhesion and, at later stages, in morphogenetic defects in the anterior neural tissues to which they mainly contribute. In addition, truncated EP-cadherin mRNA produced a duplication of the dorso-posterior axis in a significant number of cases. The expression of truncated E- and EP-cadherins in blastomeres involved in gastrulation and neural induction (B1B2 and C1), led to the duplication of the dorso-posterior axis as well as to defects in anterior structures. Morphogenetic defects obtained with truncated EP-cadherin were more severe than those induced with truncated E-cadherin. Cells derived from blastomeres injected with truncated EP-cadherin mRNA, dispersed more readily at the blastula and gastrula stages than the cells derived from the blastomeres expressing truncated E-cadherin. Presumptive mesodermal cells expressing truncated cadherins did not engage in coherent directional migration. The alteration of cadherin-mediated cell adhesion led directly to the perturbation of the convergent-extension movements during gastrulation as shown in the animal cap assays and indirectly to perturbation of neural induction. Although the cytoplasmic domains of type I cadherins share a high degree of sequence identity, the over-expression of their cytoplasmic domains induces a distinct pattern of perturbations, strongly suggesting that in vivo, each cadherin may transduce a specific adhesive signal. These graded perturbations may in part result from the relative ability of each cadherin cytoplasmic domain to titer the beta-catenin.

Cloning of a full-length cDNA encoding the neutrophil-activating peptide ENA-78 from human platelets

Here we describe the cloning of a full-length cDNA encoding a neutrophil chemoattractant peptide, ENA-78, from human platelets. The cDNA encodes a predicted sequence of 114 amino acids and contains the Cys motif C-X-C found in other members of the α-chemokine family which also includes interleukin 8 (IL-8). ENA-78 has a high degree of sequence identity with other platelet-derived chemokines which also share overlapping chemotactic activities such as GROα and the neurophil-activating peptide 2 (NAP-2; derived by proteolytic cleavage of the connective-tissue-activating peptide III (CTAP-III) ).

Prosomatostatin processing in pituitary GH3 cells. Identification and secretion of the intact propeptide.

Preprosomatostatin (preproSRIF) is a peptide hormone precursor that undergoes tissue-specific processing at either a single set of paired basic residues to yield SRIF-14 or, alternatively, at a monobasic site to produce SRIF-28, an NH2 terminally extended form of SRIF-14. Mammalian preproSRIFs are a family of precursors that are remarkably conserved from rat to humans. In five species, the signal peptide and propeptides are approximately 96% identical; this high degree of sequence identity may be indicative of functional conservation. Since the propeptide is approximately five times larger than SRIF-14, we hypothesized that it would be secreted as a separate polypeptide following proSRIF proteolytic processing. To test this idea, we raised polyclonal antibodies to the entire propeptide to follow its biosynthesis and secretion. Here we demonstrate that in transfected rat anterior pituitary GH3 cells both SRIF-14 and the intact 9.5-kDa propeptide were processed coordinately from proSRIF with identical kinetics. Treatment of the cells with chloroquine, a weak base which inhibits processing to mature SRIF-14, also inhibited the appearance of the 9.5-kDa propeptide. Approximately 40% of the propeptide was targeted to the regulated secretory pathway as determined by its quantitative secretion in response to secretagogues. We also examined the secretion of the SRIF propeptide independently of SRIF-14 by expressing a truncated "propeptide" in which SRIF-14 was deleted. Significantly, the SRIF propeptide was itself efficiently transported through the secretory pathway and secreted into the culture medium. This suggests that the propeptide possesses all the topogenic information necessary for intracellular transport. The coordinate secretion of the intact propeptide with mature SRIF-14 suggests that it might function as a novel bioactive peptide.

Discrete structural domains determine differential endoplasmic reticulum to Golgi transit times for glucose transporter isoforms.

The rate of movement of the glucose transporter isoforms Glut1 and Glut4 from the endoplasmic reticulum (ER) to the Golgi apparatus was investigated by pulse labeling and monitoring endoglycosidase H resistance in mRNA-injected Xenopus oocytes and in 3T3-L1 adipocytes, a cell line that naturally expresses both transporter isoforms. Despite their high degree of sequence identity, Glut1 and Glut4 exhibited dramatically different transit times. The t1/2 values for ER to Golgi transit for Glut1 and Glut4 were < 1 and 24 h, respectively, in oocytes and approximately 5 and 20 min, respectively, in 3T3-L1 adipocytes. Pulse-chase in conjunction with sucrose density gradient analysis revealed that the rate-limiting step in the ER to Golgi processing of Glut4 was exit from the ER and not retention in an early Golgi compartment. We analyzed the biosynthesis of Glut1/Glut4 chimeric transporters in Xenopus oocytes in order to determine whether specific domains in Glut1 and Glut4 were responsible for their distinct transit times. The first exofacial glycosylated loop and the cytoplasmic carboxyl-terminal domain of Glut4 were crucial for its delayed exit from the ER. The first transmembrane, the first exofacial, and the cytoplasmic COOH-terminal domains of Glut1 were largely responsible for Glut1's rapid processing in the ER. Some of the chimeric transporters were not fully processed. Approximately 50% of chimeric molecules containing the cytoplasmic COOH-terminal domain of Glut1 and either the first transmembrane or first exofacial domain of Glut4 were retained in early Golgi compartments and prevented from complete maturation. Normal processing of these chimeras was achieved by replacing the cytoplasmic COOH-terminal domain of Glut1 with that of Glut4. These data suggest that amino acid residues within the glycosylated exofacial loop and the cytoplasmic COOH terminus participate in a rate-limiting step in the folding of both Glut1 and Glut4 or could act as transient ER retention signals. Additionally, these results show that even chimeric molecules constructed from two highly homologous proteins can exhibit aberrant folding and post-translational processing.

Overexpression of a truncated cyclin B gene arrests Dictyostelium cell division during mitosis

A cyclin gene has been isolated from Dictyostelium discoideum and the available evidence indicates that the gene encodes a B type cyclin. The cyclin box region of the protein encoded by the gene, clb1, has the highest degree of sequence identity with the B-type cyclins of other species. Levels of cyclin B mRNA and protein oscillate during the cell cycle with maximum accumulation of mRNA occurring prior to cell division and maximum levels of protein occurring during cell division. Overexpression of a N-terminally truncated cyclin B protein lacking the destruction box inhibits cell growth by arresting cell division during mitosis. The gene is present as a single copy in the Dictyostelium genome and there is no evidence for any other highly related cyclin B genes.

Biochemical, molecular, and functional characterization of porin isoforms from potato mitochondria

The mitochondrial outer membrane of eukaryotic cells contains a voltage-dependent anion channel termed porin. In the organisms studied so far only one type of porin has been identified at the protein level. Here we present a biochemical and molecular genetic analysis of two different porin polypeptides of M(r) 34,000 and 36,000 from the outer membranes of potato mitochondria (termed POM 34 and POM 36, respectively). N-terminal sequencing and the use of labeled oligonucleotide mixtures derived from these amino acid sequences allowed the isolation of cDNA clones encoding the 34- and 36-kDa proteins. They have similar steady state protein levels and share about 75% identical amino acids suggesting that they represent isoforms. In addition, a third cDNA clone coding for a slightly different isoform of the 36-kDa protein was characterized. The polypeptides encoded by the three cDNA clones share the highest degree of sequence identity with mitochondrial porins from fungi and mammals. Tentative models of the secondary structure of the 34- and 36-kDa proteins suggest the occurrence of a 16-stranded beta-barrel typical for bacterial and mitochondrial porins. Purification of the 34-kDa protein by hydroxyapatite chromatography allowed conductance measurements in artificial bilayers. The 34-kDa protein is a voltage-dependent, channel-forming component with single channel conductances of 3.5 and 2.0 nanosiemens in 1 M KCl. In spite of the striking functional similarities to mitochondrial porins from other organisms neither the 34- nor the 36-kDa proteins are able to complement the respiratory defect of a yeast por- mutant.

Genes for mast-cell serine protease and their molecular evolution.

Trypsin-related serine proteases are encoded by a very large gene family in mammals. We describe here a comparative analysis of the genomic DNA sequences of mouse, rat, and human mast-cell-specific serine protease genes. Strong evidence was found for multiple exchanges of genetic information between closely related members of this gene family. The 5' regulatory regions of MMCP-1 and MMCP-L share a remarkably high degree of sequence identity (98%), starting 10 base pairs downstream of exon 1 and extending to the end of the presently sequenced region at position -1347 of the MMCP-1 gene. The remaining parts of the two genes share approximately 80% sequence identity. Evidence for at least two additional, but not so recent, exchanges was found in the 3' regions of the MMCP-4 and MMCP-L genes and in the 5' regions of the genes for MMCP-1 and MMCP-2. The 5' regulatory regions of all presently characterized mouse mast-cell-specific chymotrypsin-like serine protease genes exhibit over 88% sequence identity in the region from the transcription initiation site to approximately position -600. An exception is MMCP-5 which is the most distantly related member of this subfamily. The high degree of sequence similarities indicates a strong evolutionary homogenization of the 5' regulatory region, possibly by several gene conversion events. In addition, several insertions of genetic information have been identified in genes for mast-cell chymases and genes for T-cell granzymes. A number of these have been found to represent repetitive sequences, such as L1. The previously characterized tissue-specific enhancer element of the RMCP II gene was identified as a member of a middle repetitive sequence. A cDNA for a newly discovered pseudogene, closely related to the mouse mast cell chymases was isolated by polymerase chain reaction amplification from a mouse connective tissue-like mast cell line. The structure of this cDNA is presented. We also present the characterization of a novel spliced variant of MMCP-6 that contains an alternative 3' terminal exon (exon 6). The function of this variant, if any, is still unknown. A comparative analysis of amino acid sequence identities between different hematopoietic serine proteases shows that a high degree of sequence similarity does not always correlate with relateness in cleavage specificity. This indicates that the substrate specificity evolved with a higher evolutionary rate than the degree of overall amino acid sequence identity of these proteases.

Partial cloning and sequencing of the gene encoding the porcine T-cell receptor δ-chain constant region

The polymerase chain reaction (PCR) was utilized to clone the pig T-cell receptor ( TCR) δ-chain constant regionencoding gene ( Cδ). A cDNA was generated from total RNA preparations of normal pig peripheral blood lymphocytes (PBLs) and a miniature pig peripheral blood cell line (PBLCL 62.G4). The cDNA was used to amplify the porcine TCR Cδ gene by PCR using primers chosen by comparing other known Cδ sequences for sequence identity. Clones were sequenced and used to determine the primary structure of the porcine TCR C8 chain. A comparison of the nucleotide and deduced amino acid (aa) sequences with the known human, mouse, sheep and cattle sequences revealed that the primary structure of the pig TCR Cδ chain has been highly conserved. The immunoglobulin (Ig) domain has two conserved Cys residues and contains a high degree of sequence identity, whereas the hinge region is marked by a high level of diversity. The transmembrane and cytoplasmic regions are also highly conserved, including the presence of the two basic aa. Arg and Lys, in the transmembrane domain. Southern blot analysis has confirmed the presence of one TCR Cδ gene in the porcine genome, consistent with similar findings in other species. Thus, the successful cloning and sequencing of the porcine TCR Cδ gene should facilitate our understanding of the role of 78 T-lymphocytes in the swine immune system.

Isolation of a mRNA encoding a nucleoside diphosphate kinase from tomato that is up-regulated by wounding

A cDNA clone (TAB2) encoding a nucleoside diphosphate (NDP) kinase has been isolated from a tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) cDNA library. The clone is 590 bp long and exhibits a high degree of sequence identity with spinach NDP kinases I and II, Pisum sativum NDP kinase I, Arabidopsis thaliana NDP kinase, Drosophila melanogaster NDP kinase, Dictyostelium discoideum NDP kinase and human Nm 23-H1 and Nm23-H2. Northern analysis has revealed that the mRNA encoded by TAB2 is up-regulated in both leaf and stem tissue in response to wounding. The increase is apparent within 1 h of wounding and is not further elevated by application of ethylene. Southern blot analysis indicates that TAB2 is a member of a small gene family.

RDlx, a Novel Distal-less-like Homeoprotein Is Expressed in Developing Cartilages and Discrete Neuronal Tissues

From a rat chondrosarcoma we isolated a cDNA that encodes a novel homeoprotein rDlx. The homeodomain of rDlx shows a high degree of sequence identity with those of Drosophila Distal-less, mouse Dlx, and Xenopus Xdll proteins. Northern hybridization of rDlx revealed a 1.4- to 1.6-kb RNA species in a rat chondrosarcoma and a cell line derived from this tumor and in mouse C3H10T1/2 cells, but no rDlx RNA was detected in mouse NIH3T3 fibroblasts, rat skin fibroblasts, mouse C2 myoblasts, mouse myeloma S194 cells, human B-cell lymphoma Daudi cells, or human acute myelocytic leukemia cells. RNase protection assays showed that rDlx transcripts were present at high levels in 14-day-old rat embryos, 18-day-old rat embryo skeletal tissues, and adult rat brain. rDlx RNAs were present at lower levels in newborn rat rib cartilage, 18-day-old rat embryo soft tissues, newborn rat skin, and adult rat heart. rDlx transcripts were not detected in adult rat liver, spleen, lung, kidney, testis, or skeletal muscle. In situ hybridization of rat embryos at different stages revealed that rDlx transcripts were present in otic vesicle, branchial arches, apical ectodermal ridge of limb bud, developing cartilages, perichondria of mature cartilages, mesenchymal cells of developing membranous bones, developing teeth, ganglionic eminence of the telencephalon, diencephalon, olfactory epithelia, and epidermis of the skin. rDlx RNAs were also detected in the developing parasympathetic mesenteric ganglia of the gastrointestinal tract. Hence, rDlx RNAs are mainly expressed in several neuronal tissues and developing skeletal tissues.

PH DEPENDENCE OF THE ABSORPTION SPECTRA AND PHOTOCHEMICAL TRANSFORMATIONS OF THE ARCHAERHODOPSINS

Two strains of archaebacteria have been found to contain light-driven proton pumping pigments analogous to bacteriorhodopsin (bR) in Halobacterium salinarium. These proteins are called archaerhodopsin-1 (aR-1) and archaerhodopsin-2 (aR-2). Their high degree of sequence identity with bR within the putative proton channel enables us to draw some conclusions about the roles of regions where differences in amino acids exist, and in particular the surface residues, on the structure and function of retinal-based proton pumps. We have characterized the spectral and photochemical properties of these two proteins and compared them to the corresponding properties of bR. While there are some differences in absorbance maxima and kinetics of the photocycle, most of the properties of aR-1 and aR-2 are similar to those of bR. The most striking differences of these proteins with bR are the lack of an alkaline-induced red-shifted absorption species and a dramatic (apparent) decrease in the light-induced transient proton release. In membrane sheet suspensions of aR-1 at 0.15 M KCl, the order of proton release and uptake appears opposite that of bR, in which proton release precedes uptake. The nature of this behavior appears to be due to differences in the amino acid sequence at the surfaces of the proteins. In particular, the residue corresponding to the lysine at position 129 of the extracellular loop region of bR is a histidine in aR-1 and could regulate the efficient release of protons into solution in bR.

Molecular Cloning and Expression of the Gene Encoding Human Angiotensin II Type 2 Receptor

The gene of human angiotensin II type 2 (AT 2) receptor was isolated from a genomic DNA library prepared from human placenta. The coding region of the human AT 2 receptor gene was contained in a single exon coding segment of the gene indicating an intronless structure of the coding region. The amino acid sequence of human AT 2 receptor deduced from its nucleotide sequence has 363 amino acids and shows a high degree of sequence identity to rat and mouse receptor sequences. Specific binding of [ 125I]Sar 1Ile 8-angiotensin II was demonstrated in COS-7 cells transfected with a plasmid containing the human AT 2 sequence. Scatchard analysis and ligand displacement profile were typical of the AT 2 receptor. Reverse transcription-polymerase chain reaction analysis showed that AT 2 receptor mRNA was expressed in adult uterus and pheochromocytoma.