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PubMed1981

Nucleotide and corresponding amino acid sequences encoded by alpha and beta tubulin mRNAs.

Most of the mRNA sequences coding for alpha and beta tubulin in embryonic chick brain have been determined by sequencing of cloned cDNA copies of these mRNA copies of these mRNAs. From a 1,682-base pair cDNA sequence we have deduced the entire protein sequence for beta tubulin. For alpha tubulin, all but about 38 N-terminal amino acids have been deduced from the cDNA sequence. Although tyrosine has previously been shown to be post-translationally added to the C-terminus of alpha tubulin by a specific ligase, we conclude that the primary post-translational even must be the removal, not the addition of tyrosine because a terminal tyrosine is encoded by the mRNA.

Amino Acid Sequence↗
PubMed1990

Molecular cloning and expression of murine guanylate cyclase/atrial natriuretic factor receptor cDNA.

The potent diuretic and natriuretic peptide hormone atrial natriuretic factor (ANF), with vasodilatory activity also stimulates steroidogenic responsiveness in Leydig cells. The actions of ANF are mediated by its interaction with specific cell surface receptors and the membrane-bound form of guanylate cyclase represents an atrial natriuretic factor receptor (ANF-R). To understand the mechanism of ANF action in testicular steroidogenesis and to identify guanylate cyclase/ANF-R that is expressed in the Leydig cells, the primary structure of murine guanylate cyclase/ANF-R has been deduced from its cDNA sequence. A cDNA library constructed from poly(A+) RNA of murine Leydig tumor (MA-10) cell line was screened for the membrane-bound form of ANF-R/guanylate cyclase sequences by hybridization with a rat brain guanylate cyclase/ANF-R cDNA probe. The amino acid sequence deduced from the cDNA shows that murine guanylate cyclase/ANF-R cDNA consists of 1057 amino acids with 21 amino acids comprising the transmembrane domain which separates an extracellular ligand-binding domain (469 amino acid residues) and an intracellular guanylate cyclase domain (567 amino acid residues). Upon transfection of the murine guanylate cyclase/ANF-R cDNA in COS-7 cells, the expressed protein showed specific binding to 125I-ANF, stimulation of guanylate cyclase activity and production of intracellular cGMP in response to ANF. The expression of guanylate cyclase/ANF-R cDNA transfected in rat Leydig tumor cells stimulated the production of testosterone and intracellular cGMP after treatment with ANF. The results presented herein directly show that ANF can regulate the testicular steroidogenic responsiveness in addition to its known regulatory role in the control of cardiovascular homeostasis.

Amino Acid Sequence↗
PubMed1996

cDNA cloning, sequence analysis and allergological characterization of Par j 2.0101, a new major allergen of the Parietaria judaica pollen.

A clone (P2) coding for an allergen of Parietaria judaica (Pj) pollen has been isolated and sequenced from a cDNA library in lambda ZAP using a pool of 23 sera from Pj-allergic patients. The clone contained an insert of 622 nucleotides with an open reading frame of 133 amino acids (aa) and a putative signal peptide of 31 aa giving a deduced mature processed protein of 102 aa with a molecular mass of 11344 Da. The expressed recombinant protein, named rPar j 2.0101, was a major allergen since it reacted with IgE of 82% (23/28) of the sera of Pj-allergic subjects analyzed. It was shown to be a new allergen since (i) the amino acid sequence homology with the already reported recombinant allergen Par j 1.0101 was 45% and (ii) there was no cross-inhibition between rPar j 2.0101 and rPar j 1.0101. In addition, rPar j 2.0101 inhibited 35% of the specific IgE for 10-14 kDa native allergens and preincubation of sera from Pj-allergic patients with both rPar j 2.0101 and rPar j 1.0101 fully abolished the IgE recognition of the 10-14 kDa native allergen region, suggesting that these two allergens contributed to the region.

Allergens↗
PubMed2001

Human DNA repair genes.

DNA repair systems are essential for the maintenance of genome integrity. Consequently, the disregulation of repair genes can be expected to be associated with significant, detrimental health effects, which can include an increased prevalence of birth defects, an enhancement of cancer risk, and an accelerated rate of aging. Although original insights into DNA repair and the genes responsible were largely derived from studies in bacteria and yeast, well over 125 genes directly involved in DNA repair have now been identified in humans, and their cDNA sequence established. These genes function in a diverse set of pathways that involve the recognition and removal of DNA lesions, tolerance to DNA damage, and protection from errors of incorporation made during DNA replication or DNA repair. Additional genes indirectly affect DNA repair, by regulating the cell cycle, ostensibly to provide an opportunity for repair or to direct the cell to apoptosis. For about 70 of the DNA repair genes listed in Table I, both the genomic DNA sequence and the cDNA sequence and chromosomal location have been elucidated. In 45 cases single-nucleotide polymorphisms have been identified and, in some cases, genetic variants have been associated with specific disorders. With the accelerating rate of gene discovery, the number of identified DNA repair genes and sequence variants is quickly rising. This report tabulates the current status of what is known about these genes. The report is limited to genes whose function is directly related to DNA repair.

Chromosomes, Human↗
PubMed1991

Group II twintron: an intron within an intron in a chloroplast cytochrome b-559 gene.

The psbF gene of chloroplast DNAs encodes the beta-subunit of cytochrome b-559 of the photosystem II reaction center. The psbF locus of Euglena gracilis chloroplast DNA has an unusual 1042 nt group II intron that appears to be formed from the insertion of one group II intron into structural domain V of a second group II intron. Using both direct primer extension cDNA sequencing and cDNA cloning and sequencing, we have determined that a 618 nt internal intron is first excised from the 1042 nt intron of psbF pre-mRNA, resulting in a partially spliced pre-mRNA containing a 424 nt group II intron with a spliced domain V. The 424 nt intron is then removed to yield the mature psbF mRNA. Therefore, the 1042 nt intron of psbF is a group II intron within another group II intron. We use the term 'twintron' to define this new type of genetic element. Intermediates in the splicing pathway were detected by northern hybridization. Splicing of both the internal and external introns occurs via lariat intermediates. Twintron splicing was found to proceed by a sequential pathway, the internal intron being removed prior to the excision of the external intron. A possible mechanism for twintron formation by intron transposition is discussed.

Animals↗
PubMed1995

Porcine VEG proteins and tear prealbumins.

Small soluble proteins, belonging to the lipocalin family are secreted in large amounts by tongue von Ebner's glands and lachrymal glands. In humans, the lingual protein, called VEG, and the lachrymal protein, called tear prealbumin, have shown identical cDNA sequences. In the pig, we have purified homodimeric proteins with subunits of 17 kDa, both from von Ebner's glands and from lachrymal glands. In both cases, the proteins can be resolved into two isoforms on a chromatofocusing column. Partial aminoacid sequences and full cDNA sequences have been obtained for the more abundant forms purified from both tissues. The two proteins appear to be identical, as in humans. The reason why the same protein is expressed in different tissues, as well as its physiological function, still remain to be clarified.

Amino Acid Sequence↗
PubMed1994

Plasma alpha-1-antiproteinase from the Mongolian gerbil, Meriones unguiculatus: isolation, partial characterization, sequencing of cDNA, and implications for molecular evolution.

alpha-1-Antiproteinase (also called alpha-1-proteinase inhibitor or alpha-1-antitrypsin) with a molecular mass of 56 kDa was purified from plasma of the Mongolian gerbil, Meriones unguiculatus, to apparent homogeneity. It inhibited trypsin, chymotrypsin, elastase, and plasmin, but not kallikrein or thrombin. Eight cDNA clones coding for this protein were isolated from a liver cDNA library and sequenced. They contained the same coding regions consisting of a 24-residue signal peptide and a 382-residue mature protein. The reactive site sequence (P3-P3) was Val-Pro-Met-Ser-Ile-Pro, characteristic of alpha-1-antiproteinase of orthodox type [Suzuki, Y. et al. (1991) J. Biol. Chem. 266, 928-932]. A molecular phylogenetic tree of 11 orthologous inhibitors, constructed on the basis of the synonymous substitution rate, shows (i) that the reactive site region is highly conserved as compared to the other part of the molecule, which contrasts with the generally accepted view that the reactive site region of serpins is strongly hypervariable, and (ii) that the myomorphs (gerbil, rat, and two species of mouse, i.e. Mus domesticus and Mus caroli) and the caviomorph (guinea pig) fail to consist of a monophyletic order, which also contradicts the traditional taxonomy based on the morphology. In the present tree, the guinea pig joins the lagomorph (rabbit), and is rather widely separated from the myomorph branch. The result, however, supports the recent hypothesis based on the molecular evolution of several other proteins that the guinea pig does not belong to the same order as the myomorph, and the caviomorphs should be elevated in taxonomic rank and conferred an ordinal status distinct from the rodents.

Amino Acid Sequence↗
PubMed2025

Genetic analysis of four cases of Poirier Bienvenu neurodevelopmental syndrome associated with CSNK2B variant.

BACKGROUND: CSNK2B deficiency underlies the pathogenesis of Poirier-Bienvenu neurodevelopmental syndrome (POBINDS). In this study, we present four cases of pediatric seizures caused by de novo variants in CSNK2B, with the aim to reinforce the clinical and variant data pertaining to early genetic factors associated with epilepsy. METHODS: Trio whole exome sequencing were used to detect variants in the proband and her family members, and bioinformatics annotation was performed for the variant. Sanger sequencing and CSNK2B cDNA sequencing were employed to ascertain the carrier status of additional family members and evaluate the potential impact of variants on splicing. RESULTS: All four cases presented with epilepsy as the initial manifestation, accompanied by global developmental delay, particularly in language and motor developmental delay. Cases 1, 3 and 4 exhibited full-scale tonic-clonic seizures, while case 2 displayed myoclonic and typical absence seizures. Furthermore, case 2 demonstrated delayed growth and development compared to age-matched peers. No abnormality was detected in the head magnetic resonance imaging (MRI). Genetic analysis revealed novel heterozygous variants in the CSNK2B gene in all four cases, including c.175 + 1G > A, c.73-2A > G, c.291 + 1G > A and c.481delA. In case 2, reverse transcription analysis of CSNK2B mRNA revealed the retention of the 3' end sequence of Intron 2 and deletion of the 5' end sequence of Exon 3. In treatment, four case received a combination of one to three types of antiseizure medication and rehabilitation training individually. Case 1 continued to experience seizures to varying degrees, while cases 2-4 demonstrated effective seizure control. Overall motor and intellectual development improved in all four cases, however, there was slow recovery in language function. CONCLUSION: This study elucidates the molecular etiology of epilepsy in four cases with POBINDS and expands the mutational spectrum of pathogenic variants in the CSNK2B, highlighting their impact on splicing. The highly genetic heterogeneous phenotype of POBINDS relies on the detection of pathogenic variants in CSNK2B. Conventional antiseizure medication effectively control seizures, while rehabilitation treatment can significantly improve intelligence and motor function to varying degrees; however, language recovery tends to be relatively slow.

Humans↗
PubMed1980

Synthesis, cloning, and identification of DNA sequences complementary to mRNAs for alpha and beta subunits of thyrotropin.

Double-stranded cDNA sequences were synthesized, by using as templates mRNA for alpha and beta subunits of thyrotropin purified from mouse thyrotrophic pituitary tumours and cloned in Escherichia coli RR1 by insertion in the Pst I site of the bacterial plasmid pBR322 by use of poly(dA) x poly(dT) homopolymeric extensions. Plasmids containing inserted cDNA sequences were selected by resistance to tetracycline and sensitivity to ampicillin; those containing thyrotropin cDNA sequences were identified by colony hybridization with an 125I-labeled mixture of alpha and beta thyrotropin mRNAs. Plasmids carrying either alpha or beta thyrotropin cDNA sequences were further identified by hybridization to highly purified 125I-labeled alpha or beta thyrotropin mRNA, respectively. Two plasmids, one containing a 400-nucleotide alpha thyrotropin cDNA insert and the other containing a 710-nucleotide beta thyrotropin cDNA insert, were purified and characterized by restriction endonuclease digestions. Plasmid [32P]DNA containing either alpha or beta thyrotropin cDNA was then used as a hybridization probe for further characterization of alpha and beta thyrotropin mRNA from the mouse thyrotropic tumor. RNA was fractionated by agarose gel electrophoresis under denaturing and nondenaturing conditions and transferred to diazobenzyloxymethyl-paper. alpha thyrotropin mRNA of two sizes, 650 and approximately equal to 1500 nucleotides long, were identified. The larger alpha thyrotropin mRNA appeared to have marked secondary structure in its native form in contrast to the 650-nucleotide alpha thyrotropin mRNA. However, only one form of beta thyrotropin mRNA was detected with an apparent size of 710 nucleotides. We have successfully cloned and identified alpha and beta thyrotropin cDNA sequences in bacterial plasmids and used them to identify a second form of alpha thyrotropin mRNA.

Animals↗
PubMed1984

Isolation and nucleotide sequence of a full-length cDNA coding for aldolase B from human liver.

Two recombinant clones, pA2 and pA3, containing cDNA sequences for human aldolase B have been isolated from a full length human liver cDNA library. The larger one, pA3, has been subcloned in M13 phage and completely sequenced with the chain terminator method. The sequence covers 1,600 nucleotides including the whole coding region (1,050 nucleotides), 67 nucleotides from the 5' non-coding region and the whole 3' non-coding region, 440 nucleotides long, down to the poly-A tail. Comparison with rabbit aldolase A and with a partial sequence of rat aldolase B, shows a homology of about 76% for aldolase A and of about 94% for aldolase B, which indicates that the sequenced cDNA codes for the liver isoenzyme. This is the first complete sequence reported for human aldolase B. The pA3 clone strongly hybridizes to 18S mRNA from human adult liver as expected from the size of the isolated cDNA.

Amino Acid Sequence↗
PubMed1985

Human complement component C3: cDNA coding sequence and derived primary structure.

The complete cDNA coding sequence and derived amino acid sequence of human complement component C3 are presented. The encoded precursor molecule contains a signal peptide of 22 amino acid residues, the beta chain (645 residues), and the alpha chain (992 residues). The two chains are joined by four arginine residues not present in the mature protein. Several functionally important sites have been localized, such as the thiolester site, the cleavage site liberating the anaphylatoxin, and two sites of cleavage by the serine protease factor I, as well as a peptide fragment with leukocyte mobilizing activity. At least two carbohydrate attachment sites, one on each chain, have been identified. Human C3 has 79% identity to mouse C3 at the nucleotide level and 77% identity at the amino acid level. The protease alpha 2-macroglobulin and complement component C4 show considerable homology to C3, suggesting that the three proteins have evolved from a common ancestor.

Amino Acid Sequence↗
PubMed2005

[Identification and analysis of a mouse gene homologous to human hepatitis B virus pre-S1 protein-binding protein using the bioinformatics method].

OBJECTIVE: To clone and identify the mouse gene homologous to human hepatitis B virus (HBV) pre-S1 protein-binding protein (PS1BP). METHODS: The human PS1BP cDNA sequence was used as the reference sequence to search homologous mouse cDNA sequence from GenBank established by National Center for Biotechnology (NCBI), National Institute of Health (NIH), for its homologous cDNA sequences of mouse by BLASTn tool. The characteristics of mouse PS1BP protein primary structure were predicted by online software. Finally the genomic DNA structure of mouse PS1BP was deduced and compared. RESULTS: The mouse PS1BP was identified and consisted of 1455 nt, coding a protein of 484 aa. The identity of human and mouse PS1BP protein is 84.92% (411/484). The genomic DNA of mouse PS1BP consisted of 3 exons and 2 introns. CONCLUSION: The identification and characterization of mouse PS1BP cDNA and genomic DNA pave a way for further study of their structures and functions.

Amino Acid Sequence↗
PubMed2002

Prediction of the coding sequences of mouse homologues of KIAA gene: I. The complete nucleotide sequences of 100 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a human cDNA project to predict protein-coding sequences in long cDNAs (> 4 kb) since 1994. The number of these newly identified human genes exceeds 2000 and these genes are known as KIAA genes. As an extension of this project, we herein report characterization of cDNAs derived from mouse KIAA-homologous genes. A primary aim of this study was to prepare a set of mouse. KIAA-homologous cDNAs that could be used to analyze the physiological roles of KIAA genes in mice. In addition, comparison of the structures of mouse and human KIAA cDNAs might enable us to evaluate the integrity of KIAA cDNAs more convincingly. In this study, we selected mouse KIAA-homologous cDNA clones to be sequenced by screening a library of terminal sequences of mouse cDNAs in size-fractionated libraries. We present the entire sequences of 100 cDNA clones thus selected and predict their protein-coding sequences. The average size of the 100 cDNA sequences reached 5.1 kb and that of mouse KIAA-homologous proteins predicted from these cDNAs was 989 amino acid residues.

Animals↗
PubMed1988

Nucleotide sequence of cDNA clones encoding the entire precursor polypeptides for subunits IV and V of the photosystem I reaction center from spinach.

Using lambda gt11 expression cloning and immunoscreening, cDNA-containing recombinant phages for subunits IV and V of the photosystem I reaction center were isolated, sequenced and used to probe Northern blots of polyadenylated RNA prepared from spinach seedlings. The mRNA sizes for both components are approximately 1000 and 850 nucleotides, respectively. The 968 nucleotide cDNA sequence and derived amino acid sequence for subunit IV predict a single open reading frame of 231 amino acid residues (25.4 kDa). Comparison with a 13-residue N-terminal amino acid sequence determined for subunit IV suggests a mature protein of 17.3 kDa (154 residues) and a transit sequence of 77 amino acids (8.1 kDa). The corresponding data for subunit V are 677 bp (cDNA), 167 residues for the precursor protein (18.2 kDa), 98 residues for the mature polypeptide (10.8 kDa) and 69 residues for the transit peptide (7.4 kDa). Secondary structure predictions indicate that both proteins possess greatly different transit sequences and that none is membrane-spanning.

Amino Acid Sequence↗
PubMed1995

Sequence of kappa-opioid receptor cDNA in the R1.1 thymoma cell line.

To our knowledge, this is the first demonstration of kappa-opioid receptor mRNA in cells of the immune system. While the presence of opioid receptors on cells of the immune system has been controversial, cell-binding analysis has indicated that the kappa-opioid receptor is expressed by the immature T cell line R1.1. We have developed a reverse transcriptase-polymerase chain reaction protocol to amplify the mRNA extracted from R1.1 cells with primers derived from the cDNA sequence of the mouse kappa-opioid receptor. Nucleotide sequences of the amplified products were examined and two populations of cDNA were detected which differ in the 5' region upstream of the ATG start codon. Comparison of these sequences to the previously published kappa-opioid receptor cDNA sequence suggests the presence of an intron-exon junction in the 5' non-coding region.

Animals↗
PubMed1985

Nucleotide sequence of a chicken delta-crystallin gene.

We have determined the complete nucleotide sequence of one of the two non-allelic delta-crystallin genes in the chicken, arbitrarily designated delta-gene 1, using a genomic clone (lambda g delta 106) containing the entire gene sequence. By comparison of the genomic sequence and the delta-crystallin cDNA sequence previously determined, we have identified exon sequences in the genomic sequence. Thus, the presence of 17 exons and 16 introns in the gene has been clarified. The delta-crystallin polypeptide deduced from the exon sequences consists of 465 amino acids which is larger, by 19 amino acid residues, than the polypeptide deduced from the cDNA sequence previously reported. Re-examination of the cDNA sequence using the same cDNA clone previously used shows that the present exon sequences are correct and the molecular weight of the deduced delta-crystallin polypeptide is 50,615 daltons instead of the previously reported value of 48,447 daltons. In addition, some structural features of the delta-crystallin gene including putative expression signals are discussed.

Alleles↗
PubMed1995

Isolation, primary structure, and evolution of the third component of chicken complement and evidence for a new member of the alpha 2-macroglobulin family.

Although the third component of complement, C3, has been isolated and its primary structure determined from most living classes of vertebrate, limited information is available on its structure and function for aves, which represent a significant stage in complement evolution. In this study, we present the complete cDNA sequence of chicken C3, the cDNA sequences of the thioester region for two chicken alpha 2-macroglobulin (alpha 2M)-related proteins, a simplified method for purifying chicken C3, and an analysis of the C3 convertase and factor I-mediated cleavages in chicken C3. Using the reverse-transcriptase PCR, with degenerate oligonucleotide primers derived from two conserved C3 sequences (GCGEQN/TM, TWLTAY/FV) and liver mRNA as template, we isolated three distinct 220-bp PCR products, one with a high degree of sequence similarity to C3 and two to alpha 2M and pregnancy zone protein from other species. The complete cDNA sequence of chicken C3 was obtained by screening a chicken liver lambda gt10 library with the C3 PCR product and probes from the 5' end of the partial-length C3 clones. The obtained sequence is in complete agreement with the protein sequence of several tryptic peptides of purified chicken C3. Chicken pro-C3 consists of an 18-residue putative signal peptide, a 640-residue beta-chain (70 kDa), a 989-residue alpha-chain (111 kDa), and an RKRR linker region. It contains an internal thioester and three potential N-glycosylation sites, all in the alpha-chain. The convertase cleavage site, predicted to be Arg-Ser, was confirmed by sequencing the zymosan-bound C3 fragments generated upon complement activation. NH2-terminal sequencing of the purified C3 chains showed that 1) pro-C3 is indeed cleaved at the RKRR linker sequence to generate the mature two-chain molecule, and 2) the beta-chain of chicken C3 is blocked. The deduced amino acid sequence shows 54, 54, 54, 53, 52, 57, and 55% amino acid identities to human, mouse, rat, guinea pig, rabbit, cobra, and Xenopus C3, respectively, and an identity of 44, 31, and 33% to trout, hagfish, and lamprey C3, respectively. The identities to human C4, C5, and alpha 2M are 31, 29 and 23%, respectively. A phylogenetic tree for C3, C4, C5, and alpha 2M-related proteins was constructed based on the sequence data and is discussed.

Amino Acid Sequence↗
PubMed1998

Isolation and sequence of cDNA encoding the motilin precursor from monkey intestine. Demonstration of the motilin precursor in the monkey brain.

The motilin precursor cDNA has been isolated and sequenced from a cDNA library prepared from monkey small intestine. The sequence indicates a 345 bp open reading frame, a 63 bp 5' untranslated region and a 154 bp 3' untranslated region. The sequence encodes a 115 amino acid motilin precursor composed of a 25 amino acid signal peptide, the 22 amino acid motilin peptide and a 68 amino acid motilin associated peptide (MAP). Compared with the human motilin precursor cDNA, there are two amino acid substitutions in the signal peptide, one in motilin and four in the MAP. The presence of the motilin precursor in hypothalamus, hippocampus and cerebellum was demonstrated by RT-PCR.

Amino Acid Sequence↗