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PubMed1992

Gastrin-releasing peptide (GRP) is not mammalian bombesin. Identification and molecular cloning of a true amphibian GRP distinct from amphibian bombesin in Bombina orientalis.

On the basis of structural homology and similar biological activity, gastrin-releasing peptide (GRP) has been considered the mammalian equivalent of amphibian bombesin. In this paper we now show this to be incorrect. Chromatography of frog (Bombina orientalis) gut extracts demonstrated two peaks of bombesin-like immunoreactivity (BLI), one similar in size to GRP and one similar in size to amphibian bombesin. These peaks were purified by high pressure liquid chromatography then subjected to mass spectrometric analyses to determine molecular weights and amino acid sequence. Based on the amino acid sequence of the lower molecular weight BLI species, a mixed oligonucleotide probe was prepared and used to screen a B. orientalis stomach cDNA library. Sequence analysis showed that all hybridizing clones encoded a 155-amino acid protein homologous to the mammalian GRP precursor. The mass spectra of the high and low molecular weight peaks of frog gut BLI were consistent with their origin from the processing of the frog GRP (fGRP) precursor into GRP-29 and GRP-10, just like the processing of the rat GRP precursor. Sequence homology showed that the fGRP precursor is more homology showed that the fGRP precursor is more closely related to the mammalian GRP precursors than to either the frog bombesin or frog ranatensin precursors. Northern blot analysis showed that fGRP is encoded by a mRNA of 980 bases, clearly different from the 750-base mRNA which encodes frog bombesin. Northern blot analysis and in situ hybridization showed fGRP mRNA in frog brain and stomach and bombesin mRNA in frog skin, brain, and stomach. That frogs have independent genes for both GRP and bombesin raises the possibility that mammals have an as yet uncharacterized gene encoding a true mammalian bombesin.

Amino Acid Sequence↗

PubMed1990

Isolation and sequence analysis of a chalcone synthase cDNA of Matthiola incana R. Br. (Brassicaceae).

A cDNA clone (pcM12) of the chalcone synthase (CHS) of Matthiola incana R. Br. (Brassicaceae) was isolated from a cDNA library, sequenced and analysed. It comprises the complete coding sequence for the CHS and 5' and 3' untranslated regions. The deduced amino acid sequence shows that the Matthiola incana CHS consists of 394 amino acid residues. Comparison with CHS amino acid sequences of other plants indicates more than 82% homology.

Acyltransferases↗

PubMed1993

Cloning and sequence of bone morphogenetic protein 4 cDNA from fetal rat calvarial cell.

A cDNA clone encoding bone morphogenetic protein 4 (BMP 4) has been isolated from a primary fetal rat calvarial cell cDNA library. Sequencing of this clone has revealed a single open reading frame encoding a 408 amino acid protein. Comparison of 5' noncoding exon 1 portion of this cDNA with that of human bone and prostate BMP4 cDNA shows that BMP4 gene expression may possess tissue specificity.

Amino Acid Sequence↗

PubMed1993

Structure and genomic organization of immunoglobulin light chain in the channel catfish. An unusual genomic organizational pattern of segmental genes.

Channel catfish L chain cDNA was obtained through a PCR strategy and used to isolate multiple L chain clones from cDNA and genomic libraries. Sequence analysis of full-length cDNA indicates that the V region is preceded by a leader peptide, and represented by framework and CDR regions. Both VL and CL domains contain the invariant cysteines and tryptophans as well as other phylogenetically conserved L chain residues. The sequence similarity of the catfish L chain with higher vertebrate kappa- and lambda-chains, however, does not readily allow the catfish L chain to be classified. Eight cDNA clones isolated from a cDNA library were shown to represent different processed derivatives of sterile L chain transcripts. These transcripts share a similar upstream sequence region and extend downstream to include a CL or alternatively a JL segment in partial germ-line configuration that has been spliced into a CL. Sequence comparisons indicate that these transcripts represent the product of different L chain loci. Genomic Southern blot analyses with VL and CL probes indicate that there are at least 30 VL segments and at least 15 CL segments. The analysis of 17 genomic L chain clones showed that each hybridized with VL-, JL-, and CL-specific probes. Characterization of the gene segments in three of these clones indicates a previously undescribed pattern of segmental gene organization. Gene segments are found in clusters with VL, JL, and CL segments in each cluster. Within a cluster VL segments reside upstream of single copies of closely linked JL and CL segments. The proximity of VL segments downstream from JL-CL segments suggests that individual clusters may be closely linked. The VL segments are located in opposite transcriptional polarity relative to the JL and CL gene segments, which indicates that VL segments are likely rearranged to JL-CL segments by inversion rather than deletion events.

Amino Acid Sequence↗

PubMed1997

Expression of multiple alpha1-antitrypsin-like genes in hibernating species of the squirrel family.

In the chipmunk, a mammalian hibernator, a 140 kDa protein complex found in the blood, drastically decreases in concentration during hibernation. This complex contains four species of proteins, HP-20, -25, -27 and -55. In the present study, cDNA clones coding for the chipmunk HP-55 were isolated from a liver cDNA library. Sequence analysis revealed that HP-55 is produced as a precursor protein of 413 amino acids (aa), that it has a signal peptide of 24 aa, and that it contains four potential N-glycosylation sites. The deduced aa sequence shows 63% identity with that of rat alpha1-antitrypsin (alpha1-AT); however, the sequence corresponding to the reactive center P1-P1' residues was found to be Met-Leu, whereas it is Met-Ser in the rat alpha1-AT. During screening of the chipmunk liver cDNA library, four other related classes of cDNA clones were obtained, each also coding for an alpha1-AT-like protein. In spite of more than 86% overall aa sequence identity among the five chipmunk alpha1-AT-like proteins, they are highly divergent in the putative reactive center region; the putative P1-P1' sequences are Met-Leu (HP-55 or CM55-ML), Met-Met (CM55-MM), Met-Ser (CM55-MS), Ser-Ile (CM55-SI) and Ser-Thr (CM55-ST). Each of the alpha1-AT-like protein mRNAs was expressed in chipmunk liver, and the HP-55 mRNA level was greatly reduced during hibernation. Genomic Southern blot analysis and screening of a liver cDNA library from another hibernating squirrel species, the ground squirrel, also revealed expression of multiple members of the alpha1-AT gene family, whereas analysis of a cDNA library from a non-hibernating species, the tree squirrel, found only a single alpha1-AT gene.

Amino Acid Sequence↗

PubMed1987

Use of conversion adaptors to clone antigen genes in lambda gt11.

A strategy has been devised and tested to employ EcoRI conversion adaptors for cloning 5' cohesive-ended restriction fragments into the unique EcoRI site of the lambda gt11 expression vector. Five lambda gt11 chromosomal libraries were constructed with Rickettsia tsutsugamushi genomic DNA digested with restriction enzymes generating five different 5' cohesive ends. Recombinant phage yields as high as 10(7) plaque forming units were achieved without amplification of the five libraries. Sequences encoding epitopes of all eight R. tsutsugamushi polypeptide antigens, previously identified by Western blot analysis, were obtained in the five lambda gt11 expression libraries. Recombinant antigen expression was dependent on lambda gt11 lac promoter induction in 39% of the recombinants assayed. This method significantly improves the efficiency of genomic lambda gt11 library construction by eliminating blunt-ended ligation and simplifying the removal of unligated EcoRI-ended oligonucleotides.

Antigens, Bacterial↗

PubMed2026

Classification and sequencing of hepatitis D virus from a large cohort of chronically infected individuals paired with co-infecting hepatitis B virus sequencing: a genomic characterisation study.

BACKGROUND: The most severe form of viral hepatitis is caused by co-infection of hepatitis D virus (HDV) and hepatitis B virus (HBV). Phylogenetic analyses classify HBV and HDV into eight major genotypes: HBV GTA to GTH and HDV GT1 to GT8. Paired HBV and HDV sequencing data from participants with chronic hepatitis delta are scarce. We aimed to sequence and genotype HDV and HBV from a large cohort of participants from clinical studies and diverse countries of origin. METHODS: 407 participants with chronic hepatitis D from 24 countries were characterised (124 participants from MYR301 clinical trial, 93 from MYR204, 114 from MYR202, and an additional 76 participants from diverse geographical locations). HBV and HDV from participants were analysed using sequencing, enzyme immunoassay, or both to determine HBV and HDV genotypes. BLAST analysis and phylogenetics were used to determine HBV and HDV genotypes with reference sequence libraries. Bulevirtide treatment response (measured by HDV RNA decline and normalisation of alanine aminotransferase) was compared by genotype for MYR trial participants. FINDINGS: HDV sequencing assays were successful for 386 (95%) of 407 participants and HBV sequencing or serology-based HBV genotyping assays were successful for genotyping 395 (97%) participants. For individual genotypes, HBV GTD (336 [83%] participants) and HDV GT1 (364 [89%]) were the most prevalent. For paired HBV-HDV genotypes, HBV-HDV D/1 was most common (320 [79%] of 407) followed by A/1 (30 [7%]). Phylogenetic analyses of HDV full-genome sequences showed distinct clusters of sequences within HDV GT1, and four novel provisional HDV GT1 subgenotypes, HDV GT1fp to HDVGT1ip, were identified. For 218 MYR clinical trial participants, bulevirtide treatment response was similar across HDV GT1 subgenotypes (both established and newly identified). INTERPRETATION: Novel HDV subgenotypes identified in this study indicate a greater genetic diversity of HDV GT1 than previously recognised. This knowledge will be important for developing better diagnostics, and in understanding HDV genotype-specific biology and response to treatment. More extensive HDV sequencing from under-sampled regions, such as Africa, is needed to determine the true breadth of HDV sequence and genotype diversity. FUNDING: Gilead Sciences.

Hepatitis Delta Virus↗

PubMed1996

Molecular cloning and characterization of a mouse nuclear orphan receptor expressed in embryos and testes.

A mouse cDNA encoding a putative DNA-binding protein of the zinc-finger type was isolated from an E8.5 mouse embryonic cDNA library. Sequence comparison revealed a high degree of homology between this mouse cDNA and the human and rat orphan receptor Tr2-11 isolated from prostate cDNA libraries. This transcript was detected in early-to-midgestation embryos and was seen to level off during later stages of development. In adult animals, a high level of expression was detected only in the testis, starting at postnatal day 18, a stage when active meiosis begins to occur. A specific antibody was raised, and immunoreactive signal was specifically located in the adlumenal compartment of the seminiferous tubule, where advanced germ cells reside. In mice fed a vitamin A-depleted diet, where the testes were depleted of advanced germ cells, expression of this protein could not be detected, suggesting a biological relation of this orphan receptor and male germ-cell differentiation. Using a retinoic acid response element (RARE)-containing reporter system, it was demonstrated that expression of this protein dramatically repressed both the basal and the retinoic acid (RA)-regulated promoter activities of this reporter. Thus, this orphan receptor could play a role in modulating both the basic transcription machinery and the RA signalling pathway during embryogenesis and male germ-cell differentiation.

Amino Acid Sequence↗

PubMed1996

Complementary DNA cloning and sequencing of rat enteropeptidase and tissue distribution of its mRNA.

A cDNA clone encoding enteropeptidase (EC 3.4.21.9), a key enzyme for the conversion of trypsinogen to trypsin, was isolated from a rat duodenal mucosa cDNA library. Sequences of the 3585 base pair clone predicted that enteropeptidase is synthesized as a single-chain precursor form, proenteropeptidase, consisting of 1058 amino acid residues with an internal signal sequence (51 residues) and is then processed into the mature enzyme consisting of three different peptide chains, i.e., mini, light and heavy chains, not the previously reported two-chain enzyme. The structure of enteropeptidase is relatively conserved among different species and the rat enteropeptidase is 24 and 39 amino acids longer than the porcine and human ones, respectively. Northern blot analysis of rNAs from normal rat tissues revealed that the enteropeptidase mRNA of around 4.4 kb in size was expressed only in the duodenal mucosa, and high proteolytic activity of the enzyme was detected in the proximal small intestine. Additional analysis of the RNAs by RT-PCR revealed that a low level of the mRNA was also expressed in the other parts of the small intestine, i.e., jejunum and ileum. These results indicate that the biosynthesis of enteropeptidase takes place mainly in the proximal small intestine, the duodenum, and the importance of the region in the physiology of intestinal protein digestion regulated by the enzyme is suggested. Furthermore a faint signal of the mRNA was also detected in the stomach, colon and brain in which the existence of trypsin-like serine proteases were reported. The significance of the low level expression of the gene is unclear, but the potential peptide-processing function of the enzyme in these tissues is also suggested.

Amino Acid Sequence↗

PubMed1992

Cloning and sequencing of a COUP transcription factor gene expressed in Xenopus embryos.

A cDNA clone encoding COUP transcription factor, a member of the steroid/thyroid receptor superfamily, has been isolated from a Xenopus neurula (stage 17 embryo) library. Sequencing of this clone reveals an open reading frame encoding a 397 amino acid protein. The amino acid sequence of Xenopus COUP has been compared with its human and Drosophila homologues showing that there are few similarities within the amino-terminal region, whereas the remainder of the protein, including the putative DNA and ligand binding domains, is very well conserved.

Amino Acid Sequence↗

PubMed1999

Cloning of a bovine orphan transporter and its short splicing variant.

We have isolated a cDNA (bv7-3) encoding a member of the Na+,Cl(-)-dependent transporter family and its short splicing variant (bv7-3s) by screening a bovine retina cDNA library. Sequence analysis revealed that bv7-3 encodes a protein of 729 amino acids and is a bovine homologue of the rat orphan transporter v7-3-2. bv7-3s contains 265 amino acids, sharing 252 N-terminal amino acids with bv7-3. Both mRNAs for bv7-3 and bv7-3s were detected in nervous system by Northern blot analysis. In immunofluorescence analysis in transfected HEK 293T cells, myc-tagged bv7-3 was mainly detected on the plasma membrane, whereas myc-tagged bv7-3s showed a pattern of intracellular membrane staining.

Alternative Splicing↗

PubMed1998

JH8, a gene highly homologous to the mouse jerky gene, maps to the region for childhood absence epilepsy on 8q24.

Insertional inactivation of the jerky gene in transgenic mice resulted epileptic seizures, suggesting that the jerky gene was responsible for mouse epilepsy. To isolate a human homologue of the jerky gene, we screened an Expressed Sequence Tag (EST) database using the cDNA sequence of the mouse jerky gene and identified several EST clones which contained homologous sequences to mouse jerky gene. Using a clone which showed highest homology as a probe, we isolated cDNA clones from a human fetal brain cDNA library. Sequence analysis of these clones named JH8 (jerky homologue of Human on chromosome 8) indicated that it encoded a putative protein with 520 amino acid residues. The JH8 gene has 77% identity to the mouse jerky gene at the DNA level, and its protein has 76% identity and 84% similarity to the mouse protein at the amino acid level. Northern blot analysis showed that the JH8 gene is expressed ubiquitously with a major transcript of about 9.5 kb in size. Fluorescence in situ Hybridization (FISH) analysis and radiation hybrid panel mapping revealed that the JH8 gene was located on chromosome band 8q24.3 in a region that was syntenic to mouse chromosome 15, the mapping site of the mouse jerky gene. Childhood Absence Epilepsy (CAE), one type of Idiopathic Generalized Epilepsy (IGE), has been mapped to chromosome 8q24.3 by linkage analysis. These results suggest that JH8 is a strong candidate gene for CAE.

Animals↗

PubMed1992

Molecular cloning, sequence analysis and translation of proenkephalin mRNA from rat heart.

Proenkephalin mRNA is abundant in rat cardiac ventricles but surprisingly low levels of opioid peptides or precursor forms derived from proenkephalin are present in tissue extracts. Proenkephalin mRNA in rat heart was characterized at the molecular level with the use of cDNA sequencing, in vitro translation, and primer extension. Two positive proenkephalin cDNA clones were obtained by screening approx. 20,000 recombinant phages from a heart cDNA library. Sequence analysis of the cDNA clones indicated that the heart transcript was the same form as in rat brain, but differed from the germ cell-specific testis transcript that utilizes a different transcriptional start site. Heart proenkephalin cRNA translated efficiently, resulting in the synthesis of a 35 kDa protein that was immunoprecipitated by an antibody specific to the protein. The transcriptional initiation sites utilized in the heart were the same as in the brain, based on primer extension studies. These data suggest that the proenkephalin transcript found in abundance in rat heart is the same form as found in the brain, and differs from the testis-type transcript. We conclude that the scant level of proenkephalin-derived peptides in the heart is not due to an intrinsic inability of the proenkephalin transcript to translate.

Amino Acid Sequence↗

PubMed1998

Molecular characterization of higher plant NAD-dependent isocitrate dehydrogenase: evidence for a heteromeric structure by the complementation of yeast mutants.

NAD-dependent isocitrate dehydrogenase (IDH) is a key enzyme controlling the activity of the citric acid cycle. Despite more than 30 years of work, the plant enzyme remains poorly characterized. In this paper, a molecular characterization of the plant IDH is presented. Starting from probes defined according to sequence comparisons, three full-length cDNAs named Ntidha, Ntidhb and Ntidhc encoding different IDH subunits have been isolated from a Nicotiana tabacum cell suspension library. Sequence comparisons of the tobacco IDH subunits with the E. coli NADP-dependent enzyme, and the yeast IDH1 and IDH2 subunits suggested that only IDHa had the capacity to be catalytic as IDHb and IDHc were lacking certain residues implied in catalysis. The ability of antibodies raised against the recombinant IDHa protein to preferentially cross-react with IDH2 indicated that IDHa was more closely related to IDH2 than to IDH1. Complementation of yeast single IDH mutants showed that IDHb and IDHc could replace the function of the yeast regulatory IDH1 subunit. Although IDHa was unable to complement the IDH2 mutant, its catalytic function was revealed by the ability of two heteromeric enzymes, composed of either IDHa with IDHb or IDHa with IDHc, to replace IDH function in a yeast double mutant lacking both subunits. Expression studies at the protein and mRNA levels show that each subunit is present in both root and leaf tissues and that the three IDH genes respond in the same way to nitrate addition. Taken together, such observations suggest that the physiologically active enzyme is composed of the three different subunits. These results show for the first time that the plant IDH is heteromeric and that IDH subunit composition appears to be conserved between plant and animal kingdoms.

Amino Acid Sequence↗

PubMed1992

The human glutamate receptor cDNA GluR1: cloning, sequencing, expression and localization to chromosome 5.

The rat glutamate receptor is a 907 amino acid transmembrane protein. Using the rat GluR1 cDNA as a probe, we have isolated cDNA clones from a human hippocampal cDNA library. Sequence of a full length cDNA clone revealed 98.2% and 89.4% identity to the rat sequence at the amino acid and nucleotide levels respectively. The human cDNA clone detected an RNA transcript in human cerebral cortex, hippocampus and cerebellum, similar to that seen in rat. In situ hybridization experiments showed that human GluR1 mRNA is present in granule and pyramidal cells in the hippocampal formation and that there is no apparent difference of distribution between control patient and patient with Alzheimer's disease. Dot blot analysis of flow-sorted human chromosomes showed that the GluR1 gene maps to chromosome 5.

Aged↗

PubMed1994

Database on the structure of small ribosomal subunit RNA.

The database on small ribosomal subunit RNA structure contains (June 1994) 2824 nucleotide sequences. All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. The complete database is made available to the scientific community through anonymous ftp on our server in Antwerp. A special effort was made to improve electronic retrieval and a program is supplied that allows to create different file formats. The database can also be obtained from the EMBL nucleotide sequence library.

Animals↗

PubMed1997

Cloning and sequencing of a full-length sea bream (Sparus aurata) beta-actin cDNA.

A full-length cDNA clone encoding beta-actin (beta-actin) was isolated from a sea bream (Sparus aurata) liver cDNA library. Sequencing of this clone reveals an open reading frame encoding a 375 amino acid protein that shares a high degree of conservation to other known actins. The sea bream beta-actin sequence showed 98% identity to carp and human beta-actin and 95% and 94% identity to sea squirt and Dictyostelium cytoplasmic actins, respectively.

Actins↗

PubMed1998

Pyrrolidone carboxyl peptidase from the hyperthermophilic Archaeon Pyrococcus furiosus: cloning and overexpression in Escherichia coli of the gene, and its application to protein sequence analysis.

A gene for a pyrrolidone carboxyl peptidase (Pcp: EC 3.4.19.3, pyroglutamyl peptidase), which removes amino-terminal pyroglutamyl residues from peptides and proteins, has been cloned from the hyperthermophilic Archaeon Pyrococcus furiosus using its cosmid protein library, sequenced, and expressed in Escherichia coli. The DNA sequence encodes a protein containing 208 amino acid residues with methionine at the N-terminus. Analysis of the recombinant protein expressed in E. coli, including amino acid sequence analysis from the N-terminus by automated Edman degradation and ionspray mass spectrometric analysis of the peptides generated by enzymatic digestions with lysylendopeptidase and Staphylococcus aureus V8 protease, showed its primary structure to be completely identical with that deduced from its cDNA sequence. Comparison of the amino acid sequence of P. furiosus Pcp (P.f.Pcp) with those of bacterial Pcps revealed that a high degree of sequence identity (more than 40%) and conservation of the amino acid residues comprising the catalytic triad, Cys142, His166, and Glu79. On the other hand, a unique short stretch sequence (positions around 175-185) that is absent in bacterial Pcps was found in P.f.Pcp. A similar stretch has also been reported recently in the amino acid sequence of Pcp from the hyperthermophilic Archaeon Thermococcus litoralis [Littlechild et al., in abstracts of the "International Congress on Exthermophiles '98" p. 58 (1998)]. To elucidate their contribution to the hyperthermostability of these enzymes, further structural studies are required.

Amino Acid Sequence↗