Stem cDNA Library Research Articles

Sucrose partitioning between vascular bundles and storage parenchyma in the sugarcane stem: a potential role for the ShSUT1 sucrose transporter

A transporter with homology to the SUT/SUC family of plant sucrose transporters was isolated from a sugarcane (Saccharum hybrid) stem cDNA library. The gene, designated ShSUT1, encodes a protein of 517 amino acids, including 12 predicted membrane-spanning domains and a large central cytoplasmic loop. ShSUT1 was demonstrated to be a functional sucrose transporter by expression in yeast. The estimated K(m) for sucrose of the ShSUT1 transporter was 2 mM at pH 5.5. ShSUT1 was expressed predominantly in mature leaves of sugarcane that were exporting sucrose and in stem internodes that were actively accumulating sucrose. Immunolocalization with a ShSUT1-specific antiserum identified the protein in cells at the periphery of the vascular bundles in the stem. These cells became lignified and suberized as stem development proceeded, forming a barrier to apoplasmic solute movement. However, the movement of the tracer dye, carboxyfluorescein from phloem to storage parenchyma cells suggested that symplasmic connections are present. ShSUT1 may have a role in partitioning of sucrose between the vascular tissue and sites of storage in the parenchyma cells of sugarcane stem internodes.

Cloning and characterization of a ryegrass ( Lolium perenne) gene encoding cinnamoyl-CoA reductase (CCR)

Cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) catalyzes the reduction of cinnamoyl acid-CoA esters into their corresponding aldehydes, the first step in the biosynthesis of lignin. The synthesis of lignin monomers is strictly associated with the activity of this enzyme which occurs in significant concentrations in lignin producing tissues. A 1207 bp cDNA ( LpCCR) encoding a polypeptide of 344 amino acids with a predicted molecular mass of 37.4 kDa was isolated from a ryegrass ( Lolium perenne) stem cDNA library. The identity of the LpCCR was established by comparison of the deduced polypeptide sequence with other isolated plant CCR enzymes. A motif, NWYCY, representing a putative CCR active site is conserved in the encoded LpCCR amino acid sequence. The encoded polypeptide exhibits sequence similarity to CCRs from different plants, the highest identities (87 and 86%, respectively) being to CCRs from Festuca arundicinea and Hordeum vulgare. Phylogenetic analysis shows that LpCCR and CCR from other monocotyledons form a group distinct from dicot CCRs. Genomic Southern blot hybridization demonstrated that LpCCR probably is represented as a single-copy gene in the ryegrass genome. The isolation of a single genomic CCR clone ( gLpCCR) from a ryegrass leaf library further supports this observation. The gLpCCR contains four introns, the same number found in the Eucalyptus gunnii gCCR. Computer analysis of a 1333 bp 5′-flanking region of gLpCCR suggests the presence of binding sites for a MYB transcription factor and a binding site for a P-box factor of the Dof class transcription factors. In agreement with the involvement of CCR in lignification, the ryegrass CCR mRNA was detected in stem tissue only. The isolation and characterization of gLpCCR provides tools for genetically modifying plants with reduced lignin content thereby increasing forage digestibility.

Characterization of an ethylene receptor homologue from wheat and its expression during leaf senescence.

A wheat ethylene receptor homologue (W-er1) was isolated from a wheat stem cDNA library using the Arabidopsis ETR1 cDNA as a probe. The predicted amino acid sequence of W-er1 is over 70% similar to ERS1 from Arabidopsis and exhibits homology to bacterial two-component response regulators within the histidine kinase domain. Northern hybridization demonstrated that W-er1 was expressed in stem, leaf and root tissues. Treatments known to induce senescence of detached leaves including jasmonate, abscisic acid and wounding, increased the accumulation of W-er1 mRNA, while benzyladenine treatment did not. These data suggest that W-er1 may play a role in the process of leaf senescence.

Molecular cloning and characterization of a cDNA encoding a ryegrass ( Lolium perenne) ENOD40 homologue

The ENOD40 gene, found in several leguminous and non-leguminous plant species is expressed in the pericycle of legume roots early in the nodulation process, adjacent to the protoxylem poles, but before the cortical cells divide to form the nodule itself. The ENOD40 transcript contains only short open reading frames which give rise to short peptides with a signaling function. The current work reports the cloning and analysis of ENOD40 genes from perennial ryegrass (Lolium perenne) and barley (Hordeum vulgare). Specific polymerase chain reaction (PCR) techniques lead to the isolation of a 659 bp cDNA encoding an ENOD40 homologue, designated LpE-NOD40, from a Lolium perenne stem cDNA library. In addition, a partial ENOD40 cDNA of 384 bp was isolated from barley (Hordeum vulgare) by RT-PCR cloning. The LpENOD40 transcript encodes a putative dodecapeptide, similar to that identified in ENOD40s from leguminous plants and other dicots, and also to ENOD40s from monocots. The coding sequences of ryegrass and barley ENOD40 are represented by a short open reading frame of 12 amino acids. These show a high degree of similarity to each other and to other ENOD40 sequences from monocots. The corresponding genomic DNA from a genomic ryegrass lambda library revealed that the LpENOD40 gene contains no introns. Southern blot analysis shows that the ryegrass genome contains a single copy, possibly two copies, of the gene. Alignment of the ENOD40 cDNA sequences from ryegrass and barley revealed high (77%) nucleotide homology. The ENOD40 peptides are highly conserved, not only among monocots but also on comparison with the dicot peptides. The amino acid identity of region I from ryegrass with its counterparts in maize, barley and rice is 92, 83 and 75% respectively. Expression analysis by RT-PCR demonstrates that a high level of LpENOD40 gene transcript was found expressed in stem tissue, while a lower level was detected in leaves and only a very low expression in flowers of perennial ryegrass. No LpENOD40 transcript was detected in roots.

Isolation and characterization of cDNA clones encoding ADP-glucose pyrophosphorylase (AGPase) large and small subunits from chickpea (Cicer arietinum L.)

Four cDNA clones encoding two large subunits and two small subunits of the starch regulatory enzyme ADP-glucose pyrophosphorylase (AGPase) were isolated from a chickpea (Cicer arietinum L.) stem cDNA library. DNA sequence and Southern blot analyses of these clones, designated CagpL1, CagpL2 (large subunits) and CagpS1 and CagpS2 (small subunits), revealed that these isoforms represented different AGPase large and small subunits. RNA expression analysis indicated that CagpL1 was expressed strongly in leaves with reduced expression in the stem. No detectable expression was observed in seeds and roots. CagpL2 was expressed moderately in seeds followed by weak expression in leaves, stems and roots. Similar analysis showed that CagpS1 and CagpS2 displayed a spatial expression pattern similar to that observed for CagpL2 with the exception that CagpS1 showed a much higher expression in seeds than CagpS2. The spatial expression patterns of these different AGPase subunit sequences indicate that different AGPase isoforms are used to control starch biosynthesis in different organs during chickpea development.

CDNA Cloning, Characterization, and Functional Expression of Four New Monoterpene Synthase Members of the Tpsd Gene Family from Grand Fir (Abies grandis)

Grand fir (Abies grandis) is a useful model system for studying the biochemistry, molecular genetics, and regulation of defensive oleoresin formation in conifers, a process involving both the constitutive accumulation of resin (pitch) in specialized secretory structures and the induced biosynthesis of monoterpenes and sesquiterpenes (turpentine) and diterpene resin acids (rosin) by nonspecialized cells at the site of injury. A similarity-based cloning strategy, employing primers designed to conserved regions of existing monoterpene synthases and anticipated to amplify a 1000-bp fragment, unexpectedly yielded a 300-bp fragment with sequence reminiscent of a terpenoid synthase. Utilization of this amplicon as a hybridization probe afforded four new, full-length cDNA species from a wounded fir stem cDNA library that appeared to encode four distinct monoterpene synthases. Expression in Escherichia coli, followed by enzyme assay with geranyl diphosphate (C10), farnesyl diphosphate (C15) and geranylgeranyl diphosphate (C20), and analysis of the terpene products by chiral phase gas chromatography and mass spectrometry confirmed that these sequences encoded four new monoterpene synthases, including (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, and an enzyme that produces both (−)-limonene and (−)-α-pinene. The deduced amino acid sequences indicated these enzymes to be 618 to 637 residues in length (71 to 73 kDa) and to be translated as preproteins bearing an amino-terminal plastid targeting sequence of 50–60 residues. cDNA truncation to delete the transit peptide allowed functional expression of the “pseudomature” forms of these enzymes, which exhibited no change in product outcome as a result of truncation. Sequence comparison revealed that these new monoterpene synthases from grand fir are members of the Tpsd gene subfamily and resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than mechanistically related monoterpene synthases from angiosperm species. The availability of a nearly complete set of constitutive and inducible monoterpene synthases from grand fir (now numbering seven) will allow molecular dissection of the resin-based defense response in this conifer species, and detailed study of structure–function relationships among this large and diverse family of catalysts, all of which exploit the same stereochemistry in the coupled isomerization–cyclization reaction.

Molecular characterisation and expression of a wound-inducible cDNA encoding a novel cinnamyl-alcohol dehydrogenase enzyme in lucerne (Medicago sativa L.)

A lucerne (alfalfa, Medicago sativa) stem cDNA library was screened with a cinnamyl-alcohol dehydrogenase (CAD) cDNA probe from tobacco (Nicotiana tabacum cv. Samsun). Two distinctly different cDNA clones (54% identical) were isolated and identified as putative CAD-encoding cDNAs by comparison of their nucleotide sequences with those of CAD-encoding DNA sequences from other plant species. One of the cDNAs, MsaCad2, was found to be 99.4% identical at the nucleotide level to the previously isolated lucerne cad cDNA which encodes a CAD isoform involved in lignin biosynthesis. The other cDNA, MsaCad1, has not been reported previously in lucerne, and encodes a protein related to the ELI3 class of elicitor-inducible defence-related plant proteins. The MsaCad1- and MsaCad2-encoded proteins were expressed in Escherichia coli and CAD1 was shown to be active with a range of cinnamyl, benzyl and aliphatic aldehyde substrates, while CAD2 was specific for the cinnamyl aldehydes only. Each of the respective genes is present as one or two copies. The MsaCad1 gene is expressed most actively in stem and floral tissue, whereas MsaCad2 is most actively expressed in stem, hypocotyl and root tissue. In stem tissue, expression of both genes occurs predominantly in internodes 4 and 5 (from the apex). MsaCad2, in contrast to MsaCad1, is not significantly expressed in the top three internodes of the stem. Both MsaCad1 and MsaCad2 are wound-inducible, and the wound-responsiveness of each gene is modulated by salicylic acid.

Sensory and Motor Neuron-derived Factor Is a Transmembrane Heregulin That Is Expressed on the Plasma Membrane with the Active Domain Exposed to the Extracellular Environment

The beta-heregulin sensory and motor neuron-derived factor (SMDF) has been suggested to be an important regulator of Schwann cell development and proliferation. In the present study, human SMDF was expressed in cultured cell lines. The cells and the recombinant protein were used to examine the membrane association and biological activity of the growth factor. Transfection of cells with SMDF cDNA constructs bearing FLAG epitope tags at either the amino- or carboxyl-terminal ends of the polypeptide resulted in expression of anti-FLAG immunoreactive polypeptides of approximately 44 and 83 kDa. The 83-kDa polypeptide was the major form expressed on the cell surface, as demonstrated by sensitivity to proteolysis in intact cells and surface biotinylation. SMDF was tightly associated with membranes isolated from transfected cells but was solubilized by Triton X-100. Immunofluorescent staining and immunoprecipitation experiments using cells expressing amino- or carboxyl-terminal tagged SMDF revealed that only the carboxyl-terminal end of the protein is exposed on the cell surface. Membranes from SMDF-transfected cells stimulated tyrosine phosphorylation of the beta-heregulin receptor ErbB3 in Schwann cells. Conditioned medium from transfected cells contained a similar activity, suggesting that SMDF is subject to proteolytic release from the plasma membrane. In contrast with other beta-heregulin isoforms, SMDF failed to bind heparin. Stimulation of Schwann cell ErbB3 receptor phosphorylation by SMDF was not affected by inhibition of Schwann cell heparan sulfate proteoglycan synthesis. These results demonstrate that SMDF is a type II transmembrane protein. This orientation places the active epidermal growth factor homology domain, which is located near the carboxyl-terminal end of the polypeptide, on the cell surface, where it can function as a membrane-anchored growth factor.

Sesquiterpene Synthases from Grand Fir (Abies grandis): COMPARISON OF CONSTITUTIVE AND WOUND-INDUCED ACTIVITIES, AND cDNA ISOLATION, CHARACTERIZATION, AND BACTERIAL EXPRESSION OF δ-SELINENE SYNTHASE AND γ-HUMULENE SYNTHASE

Grand fir (Abies grandis) has been developed as a model system for the study of oleoresin production in response to stem wounding and insect attack. The turpentine fraction of the oleoresin was shown to contain at least 38 sesquiterpenes that represent 12.5% of the turpentine, with the monoterpenes comprising the remainder. Assays of cell-free extracts from grand fir stem with farnesyl diphosphate as substrate indicated that the constitutive sesquiterpene synthases produced the same sesquiterpenes found in the oleoresin and that, in response to wounding, only two new products were synthesized, delta-cadinene and (E)-alpha-bisabolene. A similarity based cloning strategy yielded two new cDNA species from a stem cDNA library that, when expressed in Escherichia coli and the gene products subsequently assayed, yielded a remarkable number of sesquiterpene products. The encoded enzymes have been named delta-selinene synthase and gamma-humulene synthase based on the principal products formed; however, each enzyme synthesizes three major products and produces 34 and 52 total sesquiterpenes, respectively, thereby accounting for many of the sesquiterpenes of the oleoresin. The deduced amino acid sequence of the delta-selinene synthase cDNA open reading frame encodes a protein of 581 residues (at 67.6 kDa), whereas that of the gamma-humulene synthase cDNA encodes a protein of 593 residues (at 67.9 kDa). The two amino acid sequences are 83% similar and 65% identical to each other and range in similarity from 65 to 67% and in identity from 43 to 46% when compared with the known sequences of monoterpene and diterpene synthases from grand fir. Although the two sesquiterpene synthases from this gymnosperm do not very closely resemble terpene synthases from angiosperm species (52-56% similarity and 26-30% identity, there are clustered regions of significant apparent homology between the enzymes of these two plant classes. The multi-step, multi-product reactions catalyzed by the sesquiterpene synthases from grand fir are among the most complex of any terpenoid cyclase thus far described.

Monoterpene Synthases from Grand Fir (Abies grandis): cDNA ISOLATION, CHARACTERIZATION, AND FUNCTIONAL EXPRESSION OF MYRCENE SYNTHASE, (−)-(4S)-LIMONENE SYNTHASE, AND (−)-(1S,5S)-PINENE SYNTHASE

Grand fir (Abies grandis) has been developed as a model system for studying defensive oleoresin formation in conifers in response to insect attack or other injury. The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C10) olefins in which (-)-limonene and (-)-alpha- and (-)-beta-pinene are prominent components; (-)-limonene and (-)-pinene synthase activities are also induced upon stem wounding. A similarity based cloning strategy yielded three new cDNA species from a wounded stem cDNA library that appeared to encode three distinct monoterpene synthases. After expression in Escherichia coli and enzyme assay with geranyl diphosphate as substrate, subsequent analysis of the terpene products by chiral phase gas chromatography and mass spectrometry showed that these sequences encoded a (-)-limonene synthase, a myrcene synthase, and a (-)-pinene synthase that produces both alpha-pinene and beta-pinene. In properties and reaction stereochemistry, the recombinant enzymes resemble the corresponding native monoterpene synthases of wound-induced grand fir stem. The deduced amino acid sequences indicated the limonene synthase to be 637 residues in length (73.5 kDa), the myrcene synthase to be 627 residues in length (72.5 kDa), and the pinene synthase to be 628 residues in length (71.5 kDa); all of these monoterpene synthases appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence. Sequence comparison revealed that these monoterpene synthases from grand fir resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than other monoterpene synthases from angiosperm species. This similarity between extant monoterpene, sesquiterpene, and diterpene synthases of gymnosperms is surprising since functional diversification of this enzyme class is assumed to have occurred over 300 million years ago. Wound-induced accumulation of transcripts for monoterpene synthases was demonstrated by RNA blot hybridization using probes derived from the three monoterpene synthase cDNAs. The availability of cDNA species encoding these monoterpene synthases will allow an understanding of the regulation of oleoresin formation in conifers and will ultimately permit the transgenic manipulation of this defensive secretion to enhance resistance to insects. These cDNAs also furnish tools for defining structure-function relationships in this group of catalysts that generate acyclic, monocyclic, and bicyclic olefin products.

Abietadiene Synthase from Grand Fir (Abies grandis) cDNA ISOLATION, CHARACTERIZATION, AND BACTERIAL EXPRESSION OF A BIFUNCTIONAL DITERPENE CYCLASE INVOLVED IN RESIN ACID BIOSYNTHESIS

(-)-Abietic acid, the principal diterpenoid resin acid of the wound-induced oleoresin secreted by grand fir (Abies grandis), is synthesized by the cyclization of geranylgeranyl diphosphate to (-)-abieta-7(8),13(14)-diene, followed by sequential three-step oxidation of the C-18 methyl group of the olefin to a carboxyl function. The enzyme catalyzing the cyclization reaction, abietadiene synthase, was purified from stems of wounded grand fir saplings and was digested with trypsin. Amino acid sequence information from the resulting peptides allowed construction of degenerate oligonucleotide primers, which amplified a 551-base pair fragment from a wound-induced stem cDNA library. This hybridization probe was then utilized to screen the wound-induced stem cDNA library, from which three cDNA clones were isolated that were functionally expressed in Escherichia coli, thereby confirming that a single protein catalyzes the complex, multistep cyclization of geranylgeranyl diphosphate to abietadiene. cDNA isolate Ac22.1, which yielded the highest expressed level of cyclase activity, was 2861 base pairs in length and encoded an 868-amino acid open reading frame that included a putative plastidial transit peptide. Deduced amino acid sequence comparison to other terpene cyclases revealed an amino-terminal region of the abietadiene synthase, which resembles those of enzymes that employ substrate double bond protonation to initiate the carbocationic reaction cascade, and a carboxyl-terminal region of the synthase, which resembles those of enzymes that employ ionization of the substrate allylic diphosphate ester function to initiate the cyclization reaction. This apparent fusion of segments of the two distinct terpenoid cyclase types is consistent with the novel mechanism of the bifunctional abietadiene synthase in catalyzing both protonation-initiated and ionization-initiated cyclization steps.

Cloning and sequence analysis of laccase-encoding cDNA clones from tobacco

Three laccase-encoding cDNAs were cloned from a tobacco stem cDNA library. One of them contains a full length sequence coding for a cationic laccase. The predicted polypeptide sequence shows 48% identity with sycamore laccase. Amino acid comparisons with other laccases and ascorbate oxidases have shown that this new plant laccase sequence also contains four potential copper binding regions which are highly conserved among the blue copper oxidases.

A novel receptor tyrosine phosphatase-sigma that is highly expressed in the nervous system.

A novel transmembrane receptor protein tyrosine phosphatase-sigma (RPTP-sigma) was cloned from a rat brain stem cDNA library. The extracellular segment of one form of RPTP-sigma contains 824 amino acids and is composed of three immunoglobulin-like and five fibronectin type III (FNIII)-like repeats. The 627-amino acid cytoplasmic region of RPTP-sigma consists of two catalytic domains oriented in tandem. Northern blot analyses indicate that RPTP-sigma is highly expressed in the brain as two major transcripts of 5.7 and 6.9 kilobases (kb). The 5.7-kb transcript is expressed exclusively in the brain while the 6.9-kb species can be detected in the lung and heart, but at significantly lower levels. In situ hybridization studies confirm that RPTP-sigma is localized predominantly in the nervous system and can be detected in the rat as early as embryonic day 12. During embryonic development, RPTP-sigma is expressed extensively in the central and peripheral nervous systems, including the trigeminal and dorsal root ganglia as well as the retina. In adult rat brain, expression is restricted primarily to the olfactory tubercule, cerebellum, and hippocampus. Within the latter structure, RPTP-sigma is present in the pyramidal cell layer and granular layer of the dentate gyrus. Transfection of RPTP-sigma cDNA into human embryonic kidney 293 cells results in the synthesis of a protein with an apparent molecular mass of 200 kDa as detected by immunoprecipitation and immunoblot analyses using polyclonal antibodies against the FNIII-like repeats present in the extracellular domain of RPTP-sigma. The gene for RPTP-sigma has been mapped to distal chromosome 17 in the mouse.

Identification and characterisation of cDNA clones encoding cinnamyl alcohol dehydrogenase from tobacco.

Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) is an enzyme involved in lignin biosynthesis. We have previously isolated pure CAD enzyme as two closely related polypeptides of 44 and 42.5 kDa from tobacco stems. In this paper, we report partial amino acid sequences of these two polypeptides. Based on the peptide sequences mixed oligonucleotides were used to screen a tobacco stem cDNA library and CAD cDNA clones encoding the two polypeptides were identified. DNA sequence comparisons indicate very high sequence identity between these clones both in the coding and in the 5' and 3' untranslated sequences. The close similarity between the two CAD genes leads us to suggest that they do not represent different isoforms but are the same gene from each of the two parental lines of Nicotiana tabacum cv. Samsun. Sequence comparisons with alcohol dehydrogenase 1 (ADH1) from yeast shows sequence similarities of ca. 30%, while comparisons with maize, barley and potato ADH1 sequences show similarities of not more than 23%.

Molecular cloning of a human cannabinoid receptor which is also expressed in testis

A cDNA clone encoding a receptor protein which presents all the characteristics of a guanine-nucleotide-binding protein (G-protein)-coupled receptor was isolated from a human brain stem cDNA library. The probe used (HGMP08) was a 600 bp DNA fragment amplified by a low-stringency PCR, using human genomic DNA as template and degenerate oligonucleotide primers corresponding to conserved sequences amongst the known G-protein-coupled receptors. The deduced amino acid sequence encodes a protein of 472 residues which shares 97.3% identity with the rat cannabinoid receptor cloned recently [Matsuda, Lolait, Brownstein, Young & Bronner (1990) Nature (London) 346, 561-564]. Abundant transcripts were detected in the brain, as expected, but lower amounts were also found in the testis. The same probe was used to screen a human testis cDNA library. The cDNA clones obtained were partially sequenced, demonstrating the identity of the cannabinoid receptors expressed in both tissues. Specific binding of the synthetic cannabinoid ligand [3H]CP55940 was observed on membranes from Cos-7 cells transfected with the recombinant receptor clone. In stably transfected CHO-K1 cell lines, cannabinoid agonists mediated a dose-dependent and stereoselective inhibition of forskolin-induced cyclic AMP accumulation. The ability to express the human cannabinoid receptor in mammalian cells should help in developing more selective drugs, and should facilitate the search for the endogenous cannabinoid ligand(s).

Cloning of a human cDNA expressing a high voltage‐activating. Tea‐sensitive, type‐a K+ channel which maps to chromosome 1 band p21

Over ten different mammalian genes related to the Drosophila Shaker gene (the Sh gene family) have been identified recently. These genes encode subunits of voltage-dependent K+ channels. The family consists of four subfamilies: ShI genes are homologues of Shaker; ShII, ShIII, and ShIV are homologues of three other Shaker-like genes in Drosophila, Shab, Shaw, and Shal, respectively. We report here the cloning of a human K+ channel ShIII cDNA (HKShIIIC) obtained from a brain stem cDNA library. HKShIIIC transcripts express an atypical voltage-dependent transient (A-type) K+ current in Xenopus oocytes. This current is activated by large membrane depolarizations and is extremely sensitive to the K+ channel blocker TEA unlike most A-type currents. The gene encoding HKShIIIC maps to chromosome 1p21.

CDNA cloning, primary structure, and in vitro biosynthesis of the DB58 lectin from Dolichos biflorus.

Previous studies have shown that the Dolichos biflorus plant contains a lectin in its stems and leaves, called DB58, that is closely related to the D. biflorus seed lectin. DB58 is a heterodimer composed of two closely related subunits. Immunoprecipitation of total translation products from D. biflorus stem and leaf mRNA suggests a single polypeptide precursor for both of these subunits. Several identical cDNA clones representing the entire coding region of the DB58 mRNA have been isolated from a D. biflorus stem and leaf cDNA library. The DB58 cDNA represents an mRNA encoding a polypeptide of Mr = 29,545. The predicted polypeptide is equal in length to the larger subunit of DB58 with the addition of a 22-amino acid amino-terminal signal sequence. The sequence of the DB58 lectin exhibits 84% homology to the D. biflorus seed lectin at the amino acid level, suggesting that these lectins are encoded by differentially expressed genes and may have evolved to carry out tissue-specific functions. Comparison of the DB58 sequence to other leguminous seed lectins indicates a high degree of structural conservation.

Cloning and sequence analysis of the human brain β-adrenergic receptor: Evolutionary relationship to rodent and avian β-receptors and porcine muscarinic receptors

Two cDNA clones, λ-CLFV-108 and λ-CLFV-119, encoding for the β-adrenergic receptor, have been isolated from a human brain stem cDNA library. One human genomic clone, LCV-517 (20 kb), was characterized by restriction mapping and partial sequencing. The human brain β-receptor consists of 413 amino acids with a calculated M r of 46 480. The gene contains three potential glucocorticoid receptor-binding sites. The β-receptor expressed in human brain was homology with rodent (88%) and avian (52%) β-receptors and with porcine muscarinic cholinergic receptors (31%), supporting our proposal [(1984) Proc. Natl. Acad. Sci USA 81, 272-276] that adrenergic and muscarinic cholinergic receptors are structurally related. This represents the first cloning of a neurotransmitter receptor gene from human brain.