Molecular cloning,sequencing and expression of an α2-adrenergic receptor complementary DNA from rat brain

We have isolated a cDNA clone from rat brain using a human platelet ₂-adrenergic receptor genomic clone as a probe. Comparison of the deduced amino acid sequence (450 residues) corresponding to the rat brain cDNA with that of the human platelet and human kidney ₂-adrenergic receptors showed 84% and 44% sequence similarity, respectively. The major sequence difference between the rat brain and human platelet proteins, was a stretch of 48 amino acids within the third cytosolic loop in which the similarity was only 42%. Analysis of the 48 amino acid-region indicated that the two receptors significantly differ in terms of their primary amino acid sequence and the predicted secondary and tertiary structural features. There was no sequence similarity between the human platelet and rat brain clone over the 177 bases of 3-noncoding sequence and a less than 50% similarity over a stretch of 210 nucleotides in the 5-untranslated region. Southern-blot analysis with a human platelet ₂-adrenergic receptor probe revealed the existence of a single 5.2 kb restriction fragment (KpnI/SacI) in both human and rat genomic DNA; the rat brain ₂-receptor probe, however, hybridized to a single 1.9 kb band in rat DNA. Northern-blot analysis of rat brain poly(A⁺) RNA with the rat brain cDNA probe under stringent hybridization conditions revealed a single 4.5 kb mRNA; none was detected by the human platelet receptor probe. The rat brain 4.5 kb mRNA was not detected in any (other than brain) tested rat tissues utilizing either rat brain or human platelet DNA probes. The rat brain cDNA was expressed in a mammalian cell line (COS-2A) and found to bind the ₂-adrenergic antagonist [³H]yohimbine; based on the binding-affinity for prazosin, the presently cloned receptor was pharmacologically closer to the _2A subclass. We conclude that the rat brain cDNA encodes a new ₂-adrenergic receptor subtype that may be brain-specific.Abbreviations G protein guanine nucleotide-binding proteins - cA2-47 ₂-adrenergic receptor cDNA from rat brain - SSC (1X SSC contains 0.15 M NaCl, 15 mM Na₃citrate, pH 7.0)     

RNA complementary to α-amylase mRNA in barley

Two experimental approaches demonstrate that different types of RNA complementary to -amylase mRNA are present in barley. S1 nuclease assays identify an RNA that is complementary to essentially the full length of both the type A and type B -amylase mRNAs. Complementarity, however, is imperfect: the S1 nuclease-resistant products can only be identified if they are electrophoresed as RNA-DNA hybrids. This RNA is present in developing endosperm + aleurone tissue and in mature aleurone tissue cultured in the absence of hormonal treatment or in the presence of abscisic acid, but not in shoot or root tissue. In mature aleurone tissue treated with abscisic acid, its steady-state abundance is similar to that of -amylase mRNA. Northern blot analysis indicated the presence of a second type of antisense RNA. Under conditions of moderate stringency, antisense-specific probes detect discrete hybridizing species of 1.6, 1.4, and 1.0 kilobases in mature aleurone and shoot tissues that do not represent spurious hybridization to rRNA, -amylase mRNA, or the abundant, G+C-rich mRNA for a probable amylase/protease inhibitor. The different results are consistent with the fact that the hybridization assay can tolerate relatively short regions of complementarity separated by large, nonhomologous sequences, while the nuclease protection assay cannot.     

Molecular cloning and characterization of a gibberellin-inducible, putative α-glucosidase gene from barley

A putative -glucosidase clone has been isolated from a cDNA library constructed from mRNA of barley aleurones treated with gibberellin A₃ (GA). The clone is 2752 bp in length and has an uninterrupted open reading frame encoding a polypeptide of 877 amino acids. A 680 amino acid region is 43% identical to human lysosomal -glucosidase and other glycosyl hydrolases. In isolated aleurones, the levels of the corresponding mRNA increase strongly after the application of GA, similar to the pattern exhibited by low-pI -amylase mRNA. High levels are also observed in the aleurone and scutellum after germination, while low levels are found in developing seeds. The genome contains a single form of this -glucosidase gene and two additional sequences that may be related genes or pseudogenes.Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Cloning of a cDNA complementary to rat preputial gland β-glucuronidase mRNA

Messenger RNA was isolated from rat preputial glands by guanidine HCl extraction, ethanol and salt precipitation, followed by chromatography on oligo(dT) cellulose. Double-stranded cDNA was synthesized from the mRNA and inserted into the Pst 1 site of the plasmid pBR322 by the poly(dG)·poly(dC) tailing and annealing procedure. The hybrid plasmids were used to transform $\text{E. coli}$ HB101. Recombinant clones were screened for those containing cDNA inserts complementary to β-glucuronidase mRNA by a hybridization-selection procedure. One clone, containing an insert of about 1.2 kilobases, hybridized to preputial gland mRNA which, when translated $\text{in vitro}$, gave a product that migrated with the β-glucuronidase subunit on polyacrylamide gels.     

Induction of α1-antitrypsin mRNA and cloning of its cDNA

Local inflammation was inflicted in a baboon by turpentine administration in order to induce the plasma level of α1-antitrypsin, an acute phase protein synthesized in the liver. Comparison of the α1-antitrypsin mRNA activity in the induced and non-induced baboon liver indicated that the “acute phase” response to chemical-inflicted inflammation is mediated through an increase in the steady-state level of cellular mRNA. Alpha-1-antitrypsin was then enriched from the induced baboon liver to a purity of greater than 90% by specific immunoprecipitation of polysomes. Double-stranded DNA was synthesized from the enriched mRNA and inserted into the $\text{Pst}$ I site of pBR322. Recombinant clones containing α1-antitrypsin cDNA sequences were identified by hybridselected translation and confirmed by DNA sequence analysis.     

Bovine β-crystallin complementary DNA clones: Alternating proline/alanine sequence of βB1 subunit originates from a repetitive DNA sequence

A library of recombinant plasmids carrying complementary DNA sequences synthesized from bovine lens messenger RNAs was constructed. Clones coding for five different β-crystallin subunits: βB1, βB3, βBp, βs, βA3 (and βA1), were identified by means of hybridization selection, followed by one- and two-dimensional gel electrophoresis of the translational products. Under rather stringent conditions each of these clones hybridizes with its corresponding mRNA and does not show significant cross-hybridization with mRNAs coding for other β-crystallins, except in the case of the homologous βA3 and βA1-crystalline. The βA3 and βA1 subunits seem to be encoded by one mRNA using two different AUG codons as start position for translation. We have also determined the nucleotide sequence of a βB1-crystallin cDNA (pBLβB1) which enabled us to deduce the complete amino acid sequence of the protein. The βB1-crystallin, a characteristic component of the high molecular weight crystallin aggregate (β_H), is internally homologous both at DNA and protein level as has been reported for γ-and other β-crystallins. This is in agreement with the idea that these proteins had a common ancestral precursor gene that internally duplicated. The G + C content of the coding sequence of βB1 is very high: 67% overall and even 84.2% for the first 170 nucleotides, due to a remarkable non-random codon usage. A proline/ alanine repetition in the N-terminal domain of the protein is encoded by a repetitive “simple” DNA sequence.     

Molecular cloning and primary sequence analysis of a gene encoding a putative chitinase gene in Brassica oleracea var.capitata

INTRODUCTIONPlantshavedevelopedseveralbi0chemicaldefensemechanismsinresp0nsetopath0gensandabioticstress.Fo1l0wingpathogenattack,plantsynthesizephenyl-propaniodpr0ductssuchaslignin,l0wm0l.wt.antimicrobia1comp0undsknownasphyt0alexins,andseveraldefense-relatedproteins.Amongthesepr0teinsare"pathogenesis-relatedproteins"includingthefungalcellwalldegradingenzymeschitinaseandP-1,3-glucanase[1].Endochitinasefromhigherplantscatalyzethehydr0lysis0fchitin,aP-1,4-linkedhomop0lymerofN-acetyl-D-glucos…     

Molecular cloning and primary sequence analysis of a gene encoding a putative shitinase gene in Brassica oleracea var.capitata

Chitinase,which catalyzes the hydrolysis of the β-1,4-acetyl-D-glucosamine linkages of the fungal cell wall polymer chitin,is involved in inducible plants defense system.By construction of cabbage(Brassica oleracea var. capitata) genomic library and screening the library with pRCH8,a probe of rice chitinase gene fragment,a chitinase genomic sequence was isolated.The complete uncleotide sequence of the putative cabbage chitinase gene (cabch29) was determined,with its longest open reading frame (ORF) encoding a polypeptide of 413 aa.This polypeptide consists of a 21 aa N-terminal signal peptide,two chitin-binding domains different from those of other classes of plant chitinases,and a catalytic domain.Homology analysis illustrated that this cabch29 gene has 58.8% identity at the nucleotide level with the pRCH8 ORF probe and has 50% identity at the amino acid level tiwh the catalytic domains of chitinase from bean,maize and sugar beet.Meanwhile,several kinds of cis-elements,such as TATA box,CAAT box,GATA motif,ASF-1 binding site,wound-response elements and AATAAA,have also been discovered in the flanking region of cabch29 gene.     

Location of DNA sequences complementary to small nuclear repeat RNA (fr 3-RNA) in relation to DNA sequences coding for albumin and α-fetoprotein in rat liver cells

Rat liver nuclei contain a 29-nucleotides-long RNA (fr 3-RNA) which is transcribed from middle repetitive DNA sequences. By Southern analysis of restriction fragments of rat albumin and α-fetoprotein genomic clones, DNA sequences complementary to this RNA were detected on a 4.6 kbp EcoRI fragment located 600 bp downstream from the termination exon of the albumin gene and on a 2 kbp EcoRI-HindIII fragment located 10 kbp downstream from the restriction fragment containing the α-fetoprotein site. No sequence complementary to this RNA was found either in the introns of exons of both genes or in the regions extending 7 kbp upstream from the first albumin exon and 10 kbp upstream of the first α-fetoprotein exon. We concluded that sequences complementary to fr 3-RNA are present at the 3′-end flanking regions of the rat albumin and α-fetoprotein gene complexes.     

Molecular cloning of the gene encoding α-acetolactate decarboxylase from Enterobacter aerogenes

Enterobacter aerogenes genomic library has been constructed using cosmid pJB8 in Escherichia coli. The gene encoding α-acetolactate decarboxylase (ALDC) has been isolated from this library by direct measurement of enzyme activity. The expression of the ALDC gene in E. coli appears to originate from the own promoter. Subsequent subcloning revealed that the ALDC gene locates within 1.7 kb BamHI-PstI fragment.     

Molecular cloning and sequence analysis of bovine T-cell receptor γ and δ chain genes

Nine bovine T-cell receptor (Tcr) chain (Tcrg) and three Tcr chain (Tcrd) cDNA clones were isolated from the cDNA libraries constructed from peripheral blood lymphocytes and thymocytes. Of nine Tcrg cDNA clones, only four were rearranged and contained specific V, J, and C gene segments, but the remaining five contained specific J and C or only C gene segments without the V gene segment. Three kinds of Tcrg-C, which were highly related at the nucleotide and amino acid levels, were found and designated as Tcrg-C1, Tcrg-C2, and Tcrg-C3. Compared with human and mouse Tcrg-C, bovine Tcrg-C sequences are much longer, with about 27–55 amino acids corresponding to the hinge and connector regions, where the characteristic repetitive 5-amino acid motif (TTEPP or TTKPP) exists in sheep Tcrg-C as previously reported. From three Tcrd cDNA clones, two Tcrd-V and three Tcrd-J segments were isolated. The nucleotide and deduced amino acid sequences of bovine Tcrd-C, especially the transmembrane and cytoplasmic domains, are well conserved among species. As in bovine Tcrg-C, diversity of amino acid residues in the Tcrd-C region is concentrated in the hinge regions. Southern blot analysis showed that there are at least three Tcrg-C genes and one Tcrd-C gene in the bovine genome. The analysis also revealed the presence of Tcrg-C- and Tcrd-C-associated restriction fragment length polymorphisms among bovine breeds.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers D90409-20.     

Elevated serum α-fetoprotein level of nude mice bearing hepatoma cells by treatment with monoclonal antibodies to α-fetoprotein

This study was carried out to clarify the reason for elevation of serum α-fetoprotein (AFP) level of nude mice bearing hepatoma cells after treatment with monoclonal antibodies (MoAbs) to AFP. MoAbs to AFP showed no effect on the cumulative amounts of AFP secreted from human hepatoma cell line, HuH-7, in vitro. However, the treatment of nude mice bearing HuH-7N cells (HuH-7 xenograft) with MoAbs to AFP led to elevation of the serum AFP level in spite of the fact that the growth curve of HuH-7N cells was similar to that for PBS treatment. This apparent elevation of the serum AFP level is thought to be due to the slow elimination of AFP-MoAb immune complexes with little lattice structure from circulation, but not the enhancement of AFP secretion of HuH-7N cells. Thus, when using a MoAb alone or MoAb-drug conjugate, the serum AFP level should only be cautiously used as a tumor marker for evaluating the targeting immunotherapy.     

Linkage between vitamin D-binding protein and α-fetoprotein in the mouse

The albumin gene family consists of four evolutionarily related genes that code for serum transport proteins. In rodents, the genes for albumin, |ga-fetoprotein, and |gaALB are physically linked within 100 kilobases of DNA. The fourth gene, Gc, encoding vitamin D-binding protein or group-specific component, maps to the same chromosome as the other family members, but linkage has not been established. This report describes the genetic and physical mapping of Gc in mouse and establishes that, although Gc is genetically linked to the other genes, its physical distance from them extends beyond the resolution range of yeast artificial chromosome cloning and pulsed-field gel electrophoresis. Received: 18 July 1995 / Accepted: 9 September 1995     

Unexpected role of α-fetoprotein in spermatogenesis

Background

Heat shock severely affects sperm production (spermatogenesis) and results in a rapid loss of haploid germ cells, or in other words, sperm formation (spermiogenesis) is inhibited. However, the mechanisms behind the effects of heat shock on spermatogenesis are obscure.

Methodology/Principal Findings

To identify the inhibitory factor of spermiogenesis, experimental cryptorchid (EC) mice were used in this study. Here we show that α-fetoprotein (AFP) is specifically expressed in the testes of EC mice by proteome analysis. AFP was also specifically localized spermatocytes by immunohistochemical analysis and was secreted into the circulation system of EC mice by immunoblot analysis. Since spermatogenesis of an advanced mammal cannot be reproduced with in vitro, we performed the microinjection of AFP into the seminiferous tubules of normal mice to determine whether AFP inhibits spermiogenesis in vivo. AFP was directly responsible for the block in spermiogenesis of normal mice. To investigate whether AFP inhibits cell differentiation in other models, using EC mice we performed a partial hepatectomy (PH) that triggers a rapid regenerative response in the remnant liver tissue. We also found that liver regeneration is inhibited in EC mice with PH. The result suggests that AFP released into the blood of EC mice regulates liver regeneration by inhibiting the cell division of hepatocytes.

Conclusions/Significance

AFP is a well-known cancer-specific marker, but AFP has no known function in healthy human beings. Our findings indicate that AFP expressed under EC conditions plays a role as a regulatory factor in spermatogenesis and in hepatic generation.     

The complete amino acid sequence of a mouse κ light chain

The complete amino acid sequence of the kappa-chain of the mouse myeloma protein MOPC 21 was established. The protein was reduced and alkylated with iodo[2-(14)C]acetic acid, and 21 tryptic peptides were isolated, mainly by paper electrophoresis and paper chromatography. Three large tryptic peptides (of 35, 36 and 42 residues), which were difficult to isolate in this manner, were obtained pure and in excellent yields by a combination of Sephadex G-50 gel filtration in 1% (w/v) NH(4)HCO(3) and chromatography on a DEAE-cellulose column in ammonium acetate buffer, pH8.1. Peptides overlapping the tryptic peptides were isolated from a chymotryptic digest. The chain is 214 residues long. Microheterogeneity of two peptides was observed and is believed to be due to deamidation. It was not excluded that such deamidation could occur in serum from which the protein was isolated. The sequence is compared with the sequences of two other mouse kappa-chains, and with the human kappa-chain basic sequences.     

Molecular dynamics of a DNA Holliday junction: the inverted repeat sequence d(CCGGTACCGG)₄

All-atom molecular dynamics (MD) computer simulations have been applied successfully to duplex DNA structures in solution for some years and found to give close accord with observed results. However, the MD force fields have generally not been parameterized against unusual DNA structures, and their use to obtain dynamical models for this class of systems needs to be independently validated. The four-way junction (4WJ), or Holliday junction, is a dynamic DNA structure involved in central cellular processes of homologous replication and double strand break repair. Two conformations are observed in solution: a planar open-X form (OPN) with a mobile center and four duplex arms, and an immobile stacked-X (STX) form with two continuous strands and two crossover strands, stabilized by high salt conditions. To characterize the accuracy of MD modeling on 4WJ, we report a set of explicit solvent MD simulations of ～100 ns on the repeat sequence d(CCGGTACCGG)₄ starting from the STX structure (PDB code 1dcw), and an OPN structure built for the same sequence. All 4WJ MD simulations converged to a stable STX structure in close accord with the crystal structure. Our MD beginning in the OPN form converts to the STX form spontaneously at both high and low salt conditions, providing a model for the conformational transition. Thus, these simulations provide a successful account of the dynamical structure of the STX form of d(CCGGTACCGG)₄ in solution, and provide new, to our knowledge, information on the conformational stability of the junction and distribution of counterions in the junction interior.     

DNA sequence context conceals α-anomeric lesions

DNA sequence context has long been known to modulate detection and repair of DNA damage. Recent studies using experimental and computational approaches have sought to provide a basis for this observation. We have previously shown that an α-anomeric adenosine (αA) flanked by cytosines (5'CαAC-3') resulted in a kinked DNA duplex with an enlarged minor groove. Comparison of different flanking sequences revealed that a DNA duplex containing a 5'CαAG-3' motif exhibits unique substrate properties. However, this substrate was not distinguished by unusual thermodynamic properties. To understand the structural basis of the altered recognition, we have determined the solution structure of a DNA duplex with a 5'CαAG-3' core, using an extensive set of restraints including dipolar couplings and backbone torsion angles. The NMR structure exhibits an excellent agreement with the data (total R(X) <5.3%). The αA base is intrahelical, in a reverse Watson-Crick orientation, and forms a weak base pair with a thymine of the opposite strand. In comparison to the DNA duplex with a 5'CαAC-3' core, we observe a significant reduction of the local perturbation (backbone, stacking, tilt, roll, and twist), resulting in a straighter DNA with narrower minor groove. Overall, these features result in a less perturbed DNA helix and obscure the presence of the lesion compared to the 5'CαAC-3' sequence. The improved stacking of the 5'CαAG-3' core also affects the energetics of the DNA deformation that is required to form a catalytically competent complex. These traits provide a rationale for the modulation of the recognition by endonuclease IV.