cDNA Cloning, Sequence Analysis of the Porcine LIM and Cysteine-rich Domain 1 Gene

AbstractLIM domain proteins are important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton by their interaction with various structural proteins, kinases and transcriptional regulators. Using molecular biology combined with in silico cloning, we have cloned the complete coding sequence of pig LIM and the cysteine-rich domain 1 gene (LMCD1) which encodes a 363 amino acid protein. The estimated molecular weight of the LMCD1 protein is 40,788 Da with a pI of 8.39. It was found to be highly expressed in both skeletal muscle and cardiac muscle. Alignment analysis revealed that the deduced protein sequence shares 86%, 91% and 93% homology with that of its human, mouse and rat counterparts, respectively. The LMCD1 protein was predicted by bioinformatics software to contain a novel cysteine-rich domain in the N-terminal region, two LIM domains in the C-terminal region, nine potential protein kinase C phosphorylation sites, seven casein kinase II phosphorylation sites, a tyrosine kinase phosphorylation site, seven N-glycosylation and N-myristoylation sites and a single potential N-glycosylation site, which is similar to the protein's human counterpart. Phylogenetic tree was constructed by aligning the amino acid sequences of the LIM domain from different species. In addition, four base mutations were detected by comparing the sequences of Large White pigs with those of Chinese Meishan pigs. The G294A mutation site was confirmed by polymerase chain reaction-single-strand conformation polymorphism analysis. Its allele frequencies were studied in five pig breeds.

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Characterization of the Gallate Dioxygenase Gene: Three Distinct Ring Cleavage Dioxygenases Are Involved in Syringate Degradation by Sphingomonas paucimobilis SYK-6

Journal of Bacteriology ◽

10.1128/jb.187.15.5067-5074.2005 ◽

2005 ◽

Vol 187 (15) ◽

pp. 5067-5074 ◽

Cited By ~ 51

Author(s):

Daisuke Kasai ◽

Eiji Masai ◽

Keisuke Miyauchi ◽

Yoshihiro Katayama ◽

Masao Fukuda

Keyword(s):

Amino Acid ◽

Amino Acid Sequences ◽

Terminal Region ◽

Ring Cleavage ◽

Sphingomonas Paucimobilis ◽

Acid Protein ◽

Demethylation Pathway ◽

Dioxygenase Gene ◽

Amino Acid Protein

ABSTRACT Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase β and α subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The Km for gallate and the V max were determined to be 66.9 ± 9.3 μM and 42.7 ± 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.

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A specific substrate from rabbit cerebellum for guanosine-3‘:5‘-monophosphate-dependent protein kinase. III. Amino acid sequences at the two phosphorylation sites.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)69637-7 ◽

1981 ◽

Vol 256 (7) ◽

pp. 3501-3506 ◽

Cited By ~ 3

Author(s):

A. Aitken ◽

T. Bilham ◽

P. Cohen ◽

D. Aswad ◽

P. Greengard

Keyword(s):

Amino Acid ◽

Protein Kinase ◽

Amino Acid Sequences ◽

Specific Substrate ◽

Phosphorylation Sites ◽

Dependent Protein Kinase ◽

Dependent Protein

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The substrate specificity of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle

Biochemical Journal ◽

10.1042/bj1620411 ◽

1977 ◽

Vol 162 (2) ◽

pp. 411-421 ◽

Cited By ~ 110

Author(s):

S J Yeaman ◽

P Cohen ◽

D C Watson ◽

G H Dixon

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Protein Kinase ◽

Phosphorylation Site ◽

Amino Acid Sequences ◽

Beta Subunit ◽

Alpha Subunit ◽

Phosphorylase Kinase ◽

Dependent Protein Kinase ◽

Dependent Protein

The known amino acid sequences at the two sites on phosphorylase kinase that are phosphorylated by cyclic AMP-dependent protein kinase were extended. The sequences of 42 amino acids around the phosphorylation site on the alpha-subunit and of 14 amino acids around the phosphorylation site on the beta-subunit were shown to be: alpha-subunit Phe-Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser-Glx-Pro-Asx-Gly-Gly-His-Ser-Leu-Gly-Ala-Asp-Leu-Met-Ser-Pro-Ser-Phe-Leu-Ser-Pro-Gly-Thr-Ser-Val-Phe(Ser,Pro,Gly)His-Thr-Ser-Lys; beta-subunit, Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-VALIle-Tyr-Glu-Pro-Leu-Lys. The sites on histone H2B which are phosphorylated by cyclic AMP-dependent protein kinase in vitro were identified as serine-36 and serine-32. The amino acid sequence in this region is: Lys-Lys-Arg-Lys-Arg-Ser32(P)-Arg-Lys-Glu-Ser36(P)-Tyr-Ser-Val-Tyr-Val- [Iwai, K., Ishikawa, K. & Hayashi, H. (1970) Nature (London) 226, 1056-1058]. Serine-36 was phosphorylated at 50% of the rate at which the beta-subunit of phosphorylase kinase was phosphorylated, and it was phosphorylated 6-7-fold more rapidly than was serine-32. The amino acid sequences when compared with those at the phosphorylation sites of other physiological substrates suggest that the presence of two adjacent basic amino acids on the N-terminal side of the susceptible serine residue may be critical for specific substrate recognition in vivo.

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Human liver glutathione S-transferase ψ. Chemical characterization and secondary-structure comparison with other mammalian glutathione S-transferases

Biochemical Journal ◽

10.1042/bj2430061 ◽

1987 ◽

Vol 243 (1) ◽

pp. 61-67 ◽

Cited By ~ 31

Author(s):

S V Singh ◽

A Kurosky ◽

Y C Awasthi

Keyword(s):

Amino Acid ◽

Secondary Structure ◽

Human Liver ◽

Chemical Characterization ◽

Compositional Analysis ◽

Amino Acid Sequences ◽

Terminal Region ◽

Glutathione S Transferase ◽

Structure Comparison ◽

Liver Glutathione

The isolation and chemical characterization of the anionic human liver glutathione S-transferase (GST) psi (pI 5.5) are described and compared with other GST isoenzymes reported for rat and human. Amino acid compositional analysis, substrate specificity and isoelectric focusing indicated that GST psi is a unique isoenzyme form of GST. Strikingly, however, amino acid sequence analysis of the N-terminal region indicated that GST psi was identical with GST mu in the first 23 amino acid residues reported. It is likely that these two enzyme forms are at least partially structurally related. In order to investigate further the genetic relationship of GST psi to other reported GST isoenzymes, secondary-structure analysis was performed. Despite substantial differences in the N-terminal-region amino acid sequences of some of the GST isoenzymes, the secondary structure of all the isoenzymes is highly conserved at their N-termini. The general uniformity of the secondary structure of this enzyme class at their N-termini strongly indicated that the observed diversity of these isoenzymes probably occurred as a result of a mechanism of gene duplication followed by divergence rather than a mechanism of convergent evolution.

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“Scanning mutagenesis” of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

Frontiers in Plant Science ◽

10.3389/fpls.2012.00153 ◽

2012 ◽

Vol 3 ◽

Cited By ~ 4

Author(s):

Nagib Ahsan ◽

Kirby N. Swatek ◽

Jingfen Zhang ◽

Ján A. Miernyk ◽

Dong Xu ◽

...

Keyword(s):

Amino Acid ◽

Pyruvate Dehydrogenase ◽

Pyruvate Dehydrogenase Complex ◽

Phosphorylation Site ◽

Amino Acid Sequences ◽

Dehydrogenase Complex

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Amino acid sequence of penicillopepsin. I. Isolation and characterization of the chymotryptic peptides

Canadian Journal of Biochemistry ◽

10.1139/o76-125 ◽

1976 ◽

Vol 54 (10) ◽

pp. 872-884 ◽

Cited By ~ 9

Author(s):

Alexander Kurosky ◽

Theo Hofmann

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Amino Acid Sequences ◽

Acid Protease ◽

Terminal Region ◽

Isolation And Characterization

The amino acid sequences of 48 peptides obtained from a chymotryptic digest of the mould acid protease, penicillopepsin (EC 3.4.23.7), have been determined. These peptides established the sequences of 26 unique fragments of up to 28 residues in length. The 28-residue fragment was identified as the N-terminal region. The C-terminal region is represented by a 13-residue fragment. The amino acids contained in these fragments account for some 85% of the residues of the enzyme.

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Synthetic-evolution reveals that phosphoregulation of the mitotic kinesin-5 Cin8 is constrained

10.1101/312637 ◽

2018 ◽

Cited By ~ 2

Author(s):

Alina Goldstein ◽

Darya Goldman ◽

Ervin Valk ◽

Mart Loog ◽

Liam J. Holt ◽

...

Keyword(s):

Amino Acid ◽

Phosphorylation Site ◽

Motor Domain ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

Phosphorylation Sites ◽

Wild Type ◽

Site Specific ◽

Precise Positioning ◽

Optimal Regulation

AbstractCdk1 has been found to phosphorylate the majority of its substrates in disordered regions. These phosphorylation sites do not appear to require precise positioning for their function. The mitotic kinesin-5 Cin8 was shown to be phosphoregulated at three Cdk1 sites in disordered loops within its catalytic motor domain. Here, we examined the flexibility of Cin8 phosphoregulation by analyzing the phenotypes of synthetic Cdk1-sites that were systematically generated by single amino-acid substitutions, starting from a phosphodeficient variant of Cin8. Out of 29 synthetic Cdk1 sites that we created, eight were non-functional; 19 were neutral, similar to the phosphodeficient variant; and two gave rise to phosphorylation-dependent spindle phenotypes. Of these two, one site resulted in novel phosphoregulation, and only one site, immediately adjacent to a native Cdk1 site, produced phosphoregulation similar to wild-type. This study shows that, while the gain of a single phosphorylation site can confer regulation and modulate the dynamics of the spindle, to achieve optimal regulation of a mitotic kinesin-5 motor protein, phosphoregulation has to be site-specific and precise.

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Genetic polymorphism of the extracellular region in surface associated interspersed 1.1 gene of Plasmodium falciparum field isolates from Thailand

Malaria Journal ◽

10.1186/s12936-021-03876-y ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Natpasit Chaianantakul ◽

Tippawan Sungkapong ◽

Jirapinya Changpad ◽

Keawalin Thongma ◽

Sasiwimon Sim-ut ◽

...

Keyword(s):

Amino Acid ◽

Dna Sequences ◽

Balancing Selection ◽

Haplotype Diversity ◽

Research Area ◽

Terminal Region ◽

Common Amino Acid ◽

Rich Domain ◽

Extracellular Region ◽

Variable Surface

Abstract Background A novel variable surface antigens (VSAs), Surface-associated interspersed proteins (SUFRINs), is a protein that is modified on the surface of infected red blood cell (iRBC). Modified proteins on the iRBC surface cause severe malaria, which can lead to death throughout the life cycle of a malaria parasite. Previous study suggested that SURFIN1.1 is an immunogenic membrane-associated protein which was encoded by using the surf1.1 gene expressed during the trophozoite and schizont stages. This study aimed to identify the regions of SURFIN1.1 and investigate the genetic diversity of the extracellular region of the surf1.1 gene. Methods A total of 32 blood samples from falciparum malaria cases that were diagnosed in Si Sa Ket Province, Thailand were collected. Plasmodium genomic DNA was extracted, and the extracellular region of surf1.1 gene was amplified using the polymerase chain reaction (PCR). A sequence analysis was then performed to obtain the number of haplotypes (H), the haplotype diversity (Hd), and the segregating sites (S), while the average number of nucleotide differences between two sequences (Pi); in addition, neutrality testing, Tajima’s D test, Fu and Li’s D* and F* statistics was also performed. Results From a total of 32 patient-isolated samples, 31 DNA sequences were obtained and analysed for surf1.1 gene extracellular region polymorphism. Researchers observed six distinct haplotypes in the current research area. Haplotype frequencies were 61.3%, 16.2%, and 12.9% for H1, H2, and H3, respectively. The remaining haplotype (H4-H6) frequency was 3.2% for each haplotype. Hd was 0.598 ± 0.089 with the Pi of 0.00381, and S was 15. The most common amino acid polymorphic site was E251Q; other sites included N48D, I49V, E228D, E235S, L265F, K267T, E276Q, and S288F. Fu and Li’s D* test value was − 1.24255, Fu and Li’s F* test value was − 1.10175, indicating a tendency toward negative balancing selection acting on the surf1.1 N-terminal region. The most polymorphic region was variable 2 (Var2) while cysteine-rich domain (CRD) was conserved in both the amino acid and nucleotide extracellular region of surf1.1 gene. Conclusions The Thai surf1.1 N-terminal region was well-conserved with only a few polymorphic sites remaining. In this study, the data regarding current bearing on the polymorphism of extracellular region of surf1.1 gene were reported, which might impact the biological roles of P. falciparum. In addition, may possibly serve as a suitable candidate for future development of SURFIN-based vaccines regarding malaria control. Graphic abstract

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Cloning of rodent cDNA encoding the poly(ADP-ribose) polymerase catalytic domain and analysis of mRNA levels during the cell cycle

Biochemistry and Cell Biology ◽

10.1139/o89-097 ◽

1989 ◽

Vol 67 (9) ◽

pp. 653-660 ◽

Cited By ~ 21

Author(s):

J. Thibodeau ◽

G. Gradwohl ◽

C. Dumas ◽

S. Clairoux-Moreau ◽

G. Brunet ◽

...

Keyword(s):

Cell Cycle ◽

Amino Acid ◽

Genomic Dna ◽

Catalytic Domain ◽

Binding Domain ◽

Amino Acid Sequences ◽

Phosphorylation Sites ◽

Mrna Levels ◽

C Terminus ◽

Strong Homology

We have isolated a partial 2.0 kb cDNA (pRATC) encoding the entire 489 amino acids of the NAD binding domain located at the C terminus of the rat poly(ADP-ribose) polymerase. pRATC sequences were analysed and compared with the human mRNA. Our analysis reveals a remarkable homology between the rat and human nucleotide and amino acid sequences. Although a few minor amino acid changes were detected, we have found that the total number of possible phosphorylation sites remained constant in the NAD binding domain of both enzymes. We have also found that a 102 amino acid sequence, containing the putative nucleotide binding site Gly-Lys-Gly (position 378), is perfectly conserved between the rat and human sequences. Strong homology was also detected between pRATC and genomic DNA isolated from various vertebrates. In addition, we have analysed the levels of poly(ADP-ribose) polymerase mRNA throughout the cell cycle. Our results show that the levels of mRNA culminate in the G1 phase. We have also found that the increase in enzymatic activity observed in rats following treatment with phenobarbital did not correspond to an increase in the mRNA levels.Key words: poly(ADP-ribose) polymerase.

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