scholarly journals Cloning, sequencing and expression of rat liver pyruvate carboxylase

1996 ◽  
Vol 316 (2) ◽  
pp. 631-637 ◽  
Author(s):  
Sarawut JITRAPAKDEE ◽  
Grant W. BOOKER ◽  
A. Ian CASSADY ◽  
John C. WALLACE
Keyword(s):  
Rat Liver ◽  
Pyruvate Carboxylase ◽  
Untranslated Region ◽  
Northern Analysis ◽  
Reading Frame ◽  
Liver Cdna Library ◽  
Lactating Mammary Gland ◽  
Polymerase Chain ◽  
Rapid Amplification ◽  
Sequencing And Expression

Overlapping clones encoding rat liver pyruvate carboxylase (PC) have been isolated by screening a liver cDNA library and by performing rapid amplification of cDNA ends polymerase chain reaction on total liver RNA. The sequence of rat PC cDNA contains an open reading frame of 3537 nucleotides encoding a polypeptide of 1178 amino acids with a calculated Mr of 129848. This is flanked by a 5′ untranslated region of 66 bp and a 3′ untranslated region of 421 bp including the poly(A) tail. The inferred protein sequence is 96.6% identical with mouse and 96.3% identical with human PCs, 68.4% identical with mosquito PC and 53.5% identical with yeast PC isoenzymes PC1 and PC2. On the basis of partial proteolysis and sequence homology with PC from other organisms (yeast, mosquito, mouse and human) and with other biotin enzymes, three functional domains, namely the biotin carboxylation domain, the transcarboxylation domain and the biotinyl domain, have been identified. Comparison with the known structure of the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase [Waldrop, Rayment and Holden (1994) Biochemistry 33, 10249–10256] highlights the functional importance of 11 highly conserved residues. Northern analysis revealed that PC mRNA is highly expressed in rat liver, kidney, adipose tissue and brain, moderately expressed in heart, adrenal gland and lactating mammary gland, and expressed at a low level in spleen and skeletal muscle.

Molecular cloning, characterization, and expression analysis of a novel sema-2a homologue in Polyrhachis vicina (Hymenoptera: Formicidae)

2010 ◽  
Vol 142 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Jing Luo ◽  
Geng-Si Xi ◽  
Shu-Min Lü ◽  
Ke Li ◽  
Qing Li
Keyword(s):  
Untranslated Region ◽  
Axonal Guidance ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Base Pairs ◽  
Reading Frame ◽  
Acid Protein ◽  
Polymerase Chain ◽  
Polyrhachis Vicina ◽  
Amino Acid Protein

AbstractThe semaphorin gene family plays important roles in axonal guidance in vertebrates and invertebrates. Semaphorin 2a, a member of the semaphorin family, belongs to class 2, which is found only in invertebrates. In our study, semaphorin 2a was cloned from the ant Polyrhachis vicina Roger. The full length of P. vicina semaphorin 2a (Pv-sema-2a) is 2763 base pairs (bp) and it contains a 5′-untranslated region (UTR) 92 bp long and a 3′-UTR 521 bp long. The open reading frame of Pv-sema-2a encodes a 716-amino-acid protein with a predicted molecular mass of 81.1 kilodaltons. Real-time quantitative reverse-transcription – polymerase chain reaction indicated that Pv-sema-2a mRNA is differentially expressed during P. vicina development, in the whole bodies as well as the heads of different castes. The high mRNA levels in embryos and pupae suggest that Pv-sema-2a plays an important role in ant development.

scholarly journals Molecular cloning, sequencing and expression studies of the human breast cancer cell glutaminase

2000 ◽  
Vol 345 (2) ◽  
pp. 365-375 ◽  
Author(s):  
Pedro M. GÓMEZ-FABRE ◽  
Juan C. ALEDO ◽  
Antonio DEL CASTILLO-OLIVARES ◽  
Francisco J. ALONSO ◽  
Ignacio NÚÑEZ DE CASTRO ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Human Breast Cancer ◽  
Human Liver ◽  
Molecular Size ◽  
Human Breast ◽  
Reading Frame ◽  
Rapid Amplification ◽  
Sequencing And Expression ◽  
Race Technique ◽  
Cell Growth And Proliferation

Phosphate-activated glutaminase (GA) is overexpressed in certain types of tumour but its exact role in tumour cell growth and proliferation is unknown. Here we describe the isolation of a full-length cDNA clone of human breast cancer ZR75 cells, by a combination of λgt10 cDNA library screening and the rapid amplification of cDNA ends (‘RACE’) technique. The cDNA of human GA is 2408 nt with a 1806-base open reading frame encoding a 602-residue protein with a predicted molecular mass of 66309 Da. The deduced amino acid sequence contains a putative mitochondrial import presequence of 14 residues at the N-terminal end. Heterologous expression and purification in Escherichia coli yielded a product of the expected molecular size that was recognized by using antibodies against the recombinant human GA. Sequence analyses showed that human GA was highly similar to the rat liver enzyme. Northern gel analysis revealed that the gene is present in human liver, brain and pancreas, in which a major transcript of 2.4 kb was demonstrated, but not in kidney, heart, skeletal muscle, lung or placenta. These results strongly suggest that the first human GA cloned, the GA from ZR-75 breast cancer cells, and presumably those from human liver and brain, are liver-type isoenzymes, in sharp contrast with the present view that considers the kidney type as the isoform expressed in all tissues with GA activity, with the exception of postnatal liver.

scholarly journals Folate binding protein: molecular characterization and transcript distribution in pig liver, kidney and jejunum

1996 ◽  
Vol 319 (3) ◽  
pp. 725-729 ◽  
Author(s):  
Chi Mai VAN HOOZEN ◽  
Erh-hsin LING ◽  
Charles H HALSTED
Keyword(s):  
Binding Protein ◽  
Cell Cancer ◽  
Northern Analysis ◽  
Jejunal Mucosa ◽  
Folate Binding Protein ◽  
Pig Liver ◽  
Reading Frame ◽  
Liver Cdna Library ◽  
Cancer Line ◽  
Liver And Kidney

Folate-binding protein (FBP) was identified and characterized in a pig liver cDNA library by screening with a 0.6 kb fragment from the cDNA of FBP from a human KB cell cancer line. The cDNA of pig liver FBP included 1230 bp containing 759 bp in the open reading frame with 80% similarity to the human placenta FBP. The deduced 253 amino acid sequence showed 67–73% similarity to previous sequences and contained 16 conserved cysteine residues, 11 tryptophan potential folate-binding sites, three sites for N-linked glycosylation and 14 hydrophobic C-terminal residues. Northern analysis and reverse transcriptase PCR identified transcripts in pig liver and kidney, but not in jejunal mucosa. Although defining the molecular structure of pig liver FBP, these studies suggest that this protein participates in the regulation of folate uptake by liver and kidney membranes but is not involved in folate absorption.

scholarly journals Cloning of the Self-incompatibility SFB Gene from Chinese Apricot ‘Xiaobaixing’ and Construction of the SFB Expression Vectors

2016 ◽  
Vol 141 (5) ◽  
pp. 407-413 ◽  
Author(s):  
Hai-nan Liu ◽  
Jian-rong Feng ◽  
Xiao-fang Liu ◽  
Wen-hui Li ◽  
Wen-juan Lv ◽  
...  
Keyword(s):  
Structural Characteristics ◽  
Prunus Armeniaca ◽  
Cauliflower Mosaic Virus ◽  
Expression Vectors ◽  
Variable Regions ◽  
Reading Frame ◽  
Hypervariable Regions ◽  
Polymerase Chain ◽  
Rapid Amplification ◽  
Virus Promoter

Three kinds of expression vectors of a pollen-S determinant were constructed to provide a reference for molecular breeding of self-compatible (SC) Prunus species. An S-haplotype-specific F-box (SFB) protein gene from the ‘Xiaobaixing’ apricot (Prunus armeniaca) was cloned by reverse transcription polymerase chain reaction (RT-PCR) and 3′-rapid-amplification of cDNA ends (3′-RACE). A 1136-bp sequence complementary to the 3′-end of the cDNA (GenBank accession number KP938528.2) with a 912-bp complete open reading frame (ORF) was obtained. The deduced amino acid sequence contained an F-box domain, two variable regions, and two hypervariable regions with structural characteristics similar to SFB in other Rosaceae plants. Sense, antisense, and RNA interference (RNAi) vectors for SFB were constructed by enzyme restriction. The target fragment was restricted using the corresponding restriction enzyme and then directionally inserted between the 35S cauliflower mosaic virus promoter and the nopaline synthase terminator (NOS-ter) of the expression vector pCAMBIA-35S-MCS-NOS-NPTII. The intron-containing hairpin RNA (ihpRNA) was obtained by fusion PCR. The constructed vectors were transferred into Agrobacterium tumefaciens strain LBA4404 by freezing/thawing. The RNAi vector of SFB was also transformed in tobacco (Nicotiana tabacum). The successful construction of these three expression vectors provides a basis for transforming ‘Xiaobaixing’ apricot and the breeding of SC Prunus cultivars.

Tissue distribution and regulation of 5′-deiodinase processes in lactating rats

1994 ◽  
Vol 142 (2) ◽  
pp. 205-215 ◽  
Author(s):  
L J W Jack ◽  
S Kahl ◽  
D L St Germain ◽  
A V Capuco
Keyword(s):  
Mammary Gland ◽  
Rat Liver ◽  
Messenger Rna ◽  
Northern Blotting ◽  
Type I ◽  
Mrna Levels ◽  
Lactating Mammary Gland ◽  
Liver And Kidney ◽  
Rat Mammary Gland ◽  
Polymerase Chain

Abstract Thyroxine 5′-deiodinase (5′D) catalyses deiodination of the prohormone thyroxine (T4) to the metabolically active hormone 3,5,3′-tri-iodothyronine (T3). Previously, it has been demonstrated that rat mammary gland expresses a 5′D with enzymatic properties equivalent to those of the type I enzyme (5′D-I) found in rat liver and kidney. Using complementary DNA (cDNA) for rat hepatic 5′D-I, we have examined expression of 5′D-I messenger RNA (mRNA) in liver, and mammary gland from virgin and lactating rats, and in seven other tissues from virgin rats. 5′D-I mRNA could not be detected in mammary gland either by Northern blotting or by the more sensitive technique of reverse transcribing mRNA and then amplifying the cDNA by polymerase chain reaction (RTPCR). Analysis of the seven tissues from virgin rats by RT-PCR showed 5′D-I amplicons in liver, kidney and thyroid. No amplicons were detected in adrenal gland, cardiac muscle, skeletal muscle or spleen. In addition, the effect of lactation intensity on circulating thyroid hormones, hepatic and mammary gland 5′D activity, and hepatic 5′D-I mRNA levels was examined. A strong inverse relationship was noted between increased lactation intensity (suckling burden) and circulating T4 and T3, hepatic 5′D-I activity and hepatic 5′D-I mRNA levels. Mammary gland 5′D activity was positively correlated to lactation intensity. The data presented strongly suggest that the 5′D activity expressed in lactating mammary gland is encoded by a mRNA different from the 5′D-I message found in rat liver, kidney and thyroid gland, and may help explain the differential regulation of 5′D-I activity in these organs during lactation. In addition, hepatic 5′D-I activity was found to be correlated with the concentration of 5′D-I mRNA, suggesting that regulation is pretranslational. Results are consistent with a previously suggested involvement of 5′D in establishing metabolic adaptations to support lactation. Journal of Endocrinology (1994) 142, 205–215

scholarly journals Identification and Characterization of A Lanosterol synthase Gene from Sanghuangporus Baumii

2021 ◽  
Author(s):  
XuTong Wang ◽  
TingTing Sun ◽  
Jian Sun ◽  
Zengcai Liu ◽  
Li Zou
Keyword(s):  
Untranslated Region ◽  
Mevalonate Pathway ◽  
Promoter Sequence ◽  
Mva Pathway ◽  
Reading Frame ◽  
E Coli ◽  
Lanosterol Synthase ◽  
Key Enzyme ◽  
Rapid Amplification

Abstract Lanosterol synthase (LS) is a key enzyme involved in the mevalonate pathway (MVA pathway) to produce lanosterol, which is a precursor for synthesizing Sanghuangporus baumii triterpenoids. To research the characteristics and construction of LS, LS ORF and promoter were cloned from S. baumii. A 2,445 bp S. baumii LS sequence was obtained by rapid amplification of cDNA ends (RACE) technology and recombinant PCR. S. baumii LS sequence includes a 5’-untranslated region (129 bp), a 3’-untranslated region (87 bp), and an open reading frame (2,229 bp) encoding a 734 amino acids. The molecular weight of LS is 84.99 kDa, and transcription start site of S. baumii LS promoter sequence ranged from 1 740 bp to 1790 bp. LS promoter contained 12 CAAT-boxes, 5 ABREs, 6 G-Boxes, 6 CGTCA-motifs, and so on. The S. baumii LS protein was expressed in E. coli BL21 (DE3) (84.99 kDa + 21.15 kDa tag protein). The transcription level of S. baumii LS was the highest on day 11 in mycelia (1.6-fold).

scholarly journals Molecular cloning, sequencing and characterization of cDNA to rat liver rhodanese, a thiosulphate sulphurtransferase

1991 ◽  
Vol 275 (1) ◽  
pp. 227-231 ◽  
Author(s):  
K L Weiland ◽  
T P Dooley
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Open Reading Frame ◽  
Peptide Sequence ◽  
Cdna Clones ◽  
Amino Acid Substitutions ◽  
Reading Frame ◽  
Cyanide Detoxification ◽  
Liver Cdna Library ◽  
Close Proximity

Rhodanese (EC 2.8.1.1), a mitochondrial thiosulphate sulphurtransferase, is involved in the formation of iron-sulphur complexes and cyanide detoxification. By screening a rat liver cDNA library with oligonucleotide probes complementary to portions of the published bovine rhodanese peptide sequence, rat rhodanese cDNA clones were obtained and sequenced. Comparison of the rat rhodanese cDNA open reading frame with the bovine peptide sequence demonstrated in the rat open reading frame the presence of 27 amino acid substitutions, only five of which are highly non-conservative. Thus the rat enzyme is approx. 91% identical with bovine rhodanese, or about 98% similar when conservative substitutions are considered. In addition, the rat translation product contains a Gly-Lys-Ala C-terminal tripeptide that was not observed in the bovine peptide sequence. All cysteine and proline residues are invariant between the two mammalian proteins. Computer-generated structural modelling of rat rhodanese indicated that few amino acid substitutions were present within close proximity to the active site or within the hinge region (connecting loop) between the A and B domains. Furthermore, evidence is presented showing that rhodanese is highly conserved at the DNA level among rodents, primates and a variety of other vertebrates.

Cloning and sequence analysis of complete cDNA of chitin deacetylase from Mucor racemosus

2007 ◽  
Vol 4 (2) ◽  
pp. 167-172 ◽  
Author(s):  
Jiang Xia-Yun ◽  
Zou Shu-Ming ◽  
Zhou Pei-Gen
Keyword(s):  
Amino Acid ◽  
Untranslated Region ◽  
Three Dimensional ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
Functional Domain ◽  
Mucor Racemosus ◽  
Chitin Deacetylase ◽  
Reading Frame ◽  
Rapid Amplification

AbstractA complete chitin deacetylase (CDA) complementary DNA (cDNA) from Mucor racemosus was cloned and sequenced by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification cDNA end (RACE) with gene special conserved primers. The cDNA sequence was submitted to GenBank (DQ538514). The complete cDNA with full-length of 1506 bp contained a 67 bp 5′-untranslated region, an open reading frame of 1344 bp and 95 bp 3′-untranslated region including tailing site AATAAA. The gene encoded a sequence of 448 amino acid residues and consisted of core nucleotides encoding a polysaccharide deacetylase domain covering 32% of the entire sequence. The CDA gene shared sequence homology with those of several fungi. The corresponding homology of the deduced amino acid sequences varied from 21 to 69%. Phylogenetic analysis according to the deduced amino acid sequences matched the classical fungi taxonomy. The three-dimensional structure of this protein was predicted. The protein had a whole CDA functional domain and a polysaccharide deacetylase domain.

Molecular Cloning and Cell-Free Expression of Mouse Antithrombin III

1992 ◽  
Vol 68 (03) ◽  
pp. 291-296 ◽  
Author(s):  
John K Wu ◽  
William P Sheffield ◽  
Morris A Blajchman
Keyword(s):  
Untranslated Region ◽  
Antithrombin Iii ◽  
Signal Sequence ◽  
Cdna Probe ◽  
Order Rate Constant ◽  
Free System ◽  
Reading Frame ◽  
Liver Cdna Library ◽  
At Iii ◽  
A Cell

SummaryThe homology between antithrombin III (AT-III) of mouse, of man, and that of other species was investigated. Preliminary experiments showed that mouse AT-III inhibited human α-thrombin efficiently (second order rate constant [K 2nd] 5.8 × 103 M–1 s–1) as compared to human AT-III (K 2nd 6.7 × 103 M–1), but was not recognized on immunoblots by antibodies that recognized both human and rabbit AT-III. In order to compare AT-III from different species at the molecular level, a cDNA clone for murine AT-III was isolated from a λZAP mouse liver cDNA library on the basis of hybridization to a rabbit AT-III cDNA probe. The 1509 bp murine AT-III cDNA consists of a 1398 bp open reading frame, preceded by a 15 bp 5’ untranslated region, followed by a 75 bp 3’ untranslated region. The deduced primary protein structure consists of a 32 amino acid signal sequence, with a mature portion of 433 residues. Mature murine AT-III is 89% identical to its human counterpart, 86% identical to bovine AT-III, and 82% identical to that of the rabbit. Constructs lacking the nucleotides encoding the signal sequence were engineered and expressed in a cell-free system. The resulting 47 kDa non-glycosylated translation product was capable of being cleaved by human α-thrombin, of forming SDS-stable complexes with the protease, and of binding to immobilized heparin. Isolation of the murine AT-III cDNA will make feasible molecularly defined experiments with murine AT-III in the mouse system.

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