Molecular cloning and assessment of the immunocontraceptive potential of the zona pellucida subunit 3 from Brandt's vole (Microtus brandti)

2006 ◽  
Vol 18 (3) ◽  
pp. 331 ◽  
Author(s):  
Hui Li ◽  
Yun-shang Piao ◽  
Zhi-bin Zhang ◽  
Christopher M. Hardy ◽  
Lyn A. Hinds
Keyword(s):  
Amino Acid ◽  
Zona Pellucida ◽  
Keyhole Limpet Hemocyanin ◽  
Amino Acid Residues ◽  
Igg Antibody ◽  
Reading Frame ◽  
Rapid Amplification ◽  
Ovarian Pathology ◽  
Race Pcr ◽  
Brandt’S Vole

A full-length cDNA encoding Brandt’s vole (Microtus brandti) zona pellucida glycoprotein subunit 3 (vZP3) was isolated using rapid amplification of cDNA ends–polymerase chain reaction (RACE-PCR). The cDNA contains an open reading frame of 1254 nucleotides encoding a polypeptide of 418 amino acid residues. The deduced amino acid sequence of vZP3 revealed high overall homology with hamster (82.1%), mouse (81.3%) and rat (80.6%). A synthetic vZP3 peptide corresponding to amino acid residues 328–343 was conjugated to keyhole limpet hemocyanin (KLH-vZP3328–343) and used to immunise female Brandt’s voles in order to test the efficacy of this peptide as a contraceptive antigen. High IgG antibody levels to the vZP3328–343 peptide were present in the sera of female voles immunised with KLH-vZP3328–343 and these also cross-reacted to the zona pellucida in ovaries of Brandt’s vole. The fertility of the KLH-vZP3328–343-immunised voles was reduced by 50% compared with controls without evidence of significant ovarian pathology.

Nucleotide sequence of cDNA encoding bonnet monkey (Macaca radiata) zona pellucida glycoprotein-ZP3

1995 ◽  
Vol 7 (5) ◽  
pp. 1209 ◽  
Author(s):  
SK Kolluri ◽  
R Kaul ◽  
K Banerjee ◽  
SK Gupta
Keyword(s):  
Amino Acid ◽  
Zona Pellucida ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Contraceptive Vaccine ◽  
Attachment Sites ◽  
Bonnet Monkey ◽  
Polymerase Chain ◽  
Human Use ◽  
Sugar Chains

The cDNA encoding bonnet monkey zona pellucida ZP3 from bonnet ovary has been amplified by polymerase chain reaction. The ZP3 gene has an open reading frame of 1272 nucleotides encoding a polypeptide of 424 amino acid residues which shares 93.9% overall identity with human ZP3. Bonnet ZP3 has four potential attachment sites for N-linked sugar chains which are also conserved in human ZP3. Bonnet ZP3 has 14 cysteine residues compared with 15 in human ZP3. The highest disparity between these molecules was restricted to a domain represented by amino acid residues 370-398. These results have important implications for the use of bonnet monkey as an animal model for evaluation and development of contraceptive vaccine based on ZP3 for human use.

Vector expression of adenovirus type 5 E1a proteins: evidence for E1a autoregulation

1985 ◽  
Vol 5 (10) ◽  
pp. 2684-2696
Author(s):  
D H Smith ◽  
D M Kegler ◽  
E B Ziff
Keyword(s):  
Amino Acid ◽  
Simian Virus 40 ◽  
Adenovirus Type ◽  
Simian Virus ◽  
Mutant Form ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Acid Protein ◽  
Dna Replication Origin ◽  
E1a Protein

We transiently expressed adenovirus type C E1a proteins in wild-type or mutant form from plasmid vectors which have different combinations of E1a and simian virus 40 enhancer elements and which contain the DNA replication origin of SV40 and can replicate in COS 7 cells. We measured the levels of E1a mRNA encoded by the vectors and the transition regulation properties of the protein products. Three vectors encoded equivalent levels of E1a mRNA in COS 7 cells: (i) a plasmid encoding the wt 289-amino acid E1a protein (this complemented the E1a deletion mutant dl312 for early region E2a expression under both replicative and nonreplicative conditions); (ii) a vector for the wt 243-amino acid E1a protein (this complemented dl312 weakly and only under conditions of high multiplicities of dl312); (iii) a mutant, pSVXL105, in which amino acid residues-38 through 44 of the 289-amino acid E1a protein (which includes two highly conserved residues) are replaced by 3 novel amino acids (this also complemented dl312 efficiently). A fourth vector, mutant pSVXL3 with which linker substitution shifts the reading frame to encode a truncated 70-amino acid fragment from the amino terminus of the 289-amino acid protein, was unable to complement dl312. Surprisingly, pSVXL3 overexpressed E1a mRNA approximately 30-fold in COS 7 cells in comparison with the other vectors. The pSVXL3 overexpression could be reversed by cotransfection with a wt E1a vector. We suggest that wt E1a proteins regulate the levels of their own mRNAs through the recently described transcription repression functions of the 289- and 243-amino acid E1a protein products and that pSVXL3 fails to autoregulate negatively.

scholarly journals Mutation avoidance and DNA repair proficiency in Ustilago maydis are differentially lost with progressive truncation of the REC1 gene product.

1995 ◽  
Vol 15 (10) ◽  
pp. 5329-5338 ◽  
Author(s):  
K Onel ◽  
M P Thelen ◽  
D O Ferguson ◽  
R L Bennett ◽  
W K Holloman
Keyword(s):  
Amino Acid ◽  
Mutation Rate ◽  
Spontaneous Mutation ◽  
Ustilago Maydis ◽  
Radiation Sensitivity ◽  
Open Reading Frame ◽  
Exonuclease Activity ◽  
Amino Acid Residues ◽  
Spontaneous Mutation Rate ◽  
Reading Frame

The REC1 gene of Ustilago maydis has an uninterrupted open reading frame, predicted from the genomic sequence to encode a protein of 522 amino acid residues. Nevertheless, an intron is present, and functional activity of the gene in mitotic cells requires an RNA processing event to remove the intron. This results in a change in reading frame and production of a protein of 463 amino acid residues. The 3'-->5' exonuclease activity of proteins derived from the REC1 genomic open reading frame, the intronless open reading frame, and several mutants was investigated. The mutants included a series of deletions constructed by removing restriction fragments at the 3' end of the cloned REC1 gene and a set of mutant alleles previously isolated in screens for radiation sensitivity. All of these proteins were overproduced in Escherichia coli as N-terminal polyhistidine-tagged fusions that were subsequently purified by immobilized metal affinity chromatography and assayed for 3'-->5' exonuclease activity. The results indicated that elimination of the C-terminal third of the protein did not result in a serious reduction in 3'-->5' exonuclease activity, but deletion into the midsection caused a severe loss of activity. The biological activity of the rec1-1 allele, which encodes a truncated polypeptide with full 3'-->5' exonuclease activity, and the rec1-5 allele, which encodes a more severely truncated polypeptide with no exonuclease activity, was investigated. The two mutants were equally sensitive to the lethal effect of UV light, but the spontaneous mutation rate was elevated 10-fold over the wild-type rate in the rec1-1 mutant and 100-fold in the rec1-5 mutant. The elevated spontaneous mutation rate correlated with the ablation of exonuclease activity, but the radiation sensitivity did not. These results indicate that the C-terminal portion of the Rec1 protein is not essential for exonuclease activity but is crucial in the role of REC1 in DNA damage repair.

bimA encodes a member of the tetratricopeptide repeat family of proteins and is required for the completion of mitosis in Aspergillus nidulans

1991 ◽  
Vol 99 (4) ◽  
pp. 711-719
Author(s):  
K.L. O'Donnell ◽  
A.H. Osmani ◽  
S.A. Osmani ◽  
N.R. Morris
Keyword(s):  
Amino Acid ◽  
Aspergillus Nidulans ◽  
Essential Gene ◽  
Tetratricopeptide Repeat ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Reading Frame ◽  
Integrative Transformation

The recessive, temperature-sensitive bimA1 mutation of Aspergillus nidulans blocks nuclei in metaphase at restrictive temperature. To determine whether the bimA product is essential, integrative transformation was used to create a mutation in the bimA gene. The mutation was maintained in a heterokaryon and the phenotype of spores produced by the heterokaryon was analyzed. Molecular disruption of the wild-type bimA gene is recessive in the heterokaryon and causes a metaphase block, demonstrating that bimA is an essential gene for mitosis. bimA was cloned by DNA-mediated complementation of its mutant phenotype at restrictive temperature, and the nucleotide sequence of a full-length cDNA was determined. A single large open reading frame was identified in the cDNA sequence, which predicts a protein containing 806 amino acid residues that is related (30.4% identity) to the Schizosaccharomyces pombe nuc2+ gene product, which also is required for completion of mitosis. The sequence of the bimA gene indicates that it is a member of a family of mostly nuclear proteins that contain a degenerate 34 amino acid repeat, the TPR (tetratricopeptide repeat) gene family.

Gene Cloning, Nucleotide Sequencing, and Purification and Characterization of the Low-Specificityl-Threonine Aldolase from Pseudomonas sp. Strain NCIMB 10558

1998 ◽  
Vol 64 (2) ◽  
pp. 549-554 ◽  
Author(s):  
Ji-Quan Liu ◽  
Saeko Ito ◽  
Tohru Dairi ◽  
Nobuya Itoh ◽  
Michihiko Kataoka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Significant Loss ◽  
Site Directed Mutagenesis ◽  
Nucleotide Sequencing ◽  
Predicted Amino Acid Sequence ◽  
Amino Acid Residues ◽  
Purification And Characterization ◽  
Reading Frame ◽  
Threonine Aldolase

ABSTRACT A low-specificity l-threonine aldolase (l-TA) gene from Pseudomonas sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia colicells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5′-phosphate as a coenzyme, is strictlyl specific at the α position, whereas it cannot distinguish between threo and erythro forms at the β position. In addition to threonine, the enzyme also acts on various other l-β-hydroxy-α-amino acids, includingl-β-3,4-dihydroxyphenylserine,l-β-3,4-methylenedioxyphenylserine, andl-β-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificityl-TA from Saccharomyces cerevisiae,l-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms. However, lysine 207 of low-specificity l-TA from Pseudomonas sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys207 of thel-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5′-phosphate of the enzyme to catalyze the reversible aldol reaction.

Cloning and Expression of a Novel Protease with Fibrinolytic Activity from Arenicola cristata

2014 ◽  
Vol 998-999 ◽  
pp. 210-213
Author(s):  
Chun Ling Zhao ◽  
Wen Jing Yu ◽  
Ji Yu Ju
Keyword(s):  
Amino Acid ◽  
Recombinant Protein ◽  
Active Form ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Reading Frame ◽  
Arenicola Cristata ◽  
Fibrin Plate ◽  
Serine Protease Family

cDNA of a novel protease, designated as AFEI, was cloned from digestive tract of Arenicola cristata by RACE. The cDNA of AFEIcomprised 897bp and an open reading frame that encoded polypeptides of 264 amino acid residues. AFEIshowed similarity to serine protease family and contained the conserved catalytic amino acid residues. The gene encoding the active form of AFEIwas expressed in E.coli and the purified recombinant protein could dissolve an artificial fibrin plate with plasminogen, which indicated the recombinant protein might be a plasminogen activator for thrombosis therapy.

Molecular cloning, characterization and tissue specificity of the expression in the TAC1 genes from Henan Huai goat (Capra hircus)

2019 ◽  
Author(s):  
Zhilong Tian ◽  
Yuqin Wang ◽  
Huibin Shi ◽  
Zhibo Wu ◽  
Xiaohui Zhang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Phylogenetic Analyses ◽  
Expression Patterns ◽  
Amino Acid Sequences ◽  
Full Length ◽  
Capra Hircus ◽  
Reading Frame ◽  
Relative Molecular Weight ◽  
Rapid Amplification ◽  
And Function

To further to understand the structure and function of the TAC1 gene, we cloned the full-length cDNAs of the TAC1 genes from goat by rapid amplification of cDNA ends-PCR and the qRT-PCR was used to analyze the TAC1 mRNA expression patterns of goat various tissues. The full-length cDNA of goat TAC1 was 1176 bp, with a 339 bp open reading frame encoding 112 amino acids. The amino acid sequence analysis revealed that goat TAC1 gene encoded a water-drain protein and its relative molecular weight and isoelectric point was 13,012.86 Da and 6.29 respectively. Alignment and phylogenetic analyses revealed that their amino acid sequences were highly similar to those of other vertebrates. TAC1 expression of the goat of the brain, cerebellum, medulla oblongata, heart, liver, spleen, lung, kidney, uterus, ovaries. These results serve as a foundation for further study on the Capra hircus TAC1 gene.

scholarly journals Replication Specificity Elements of the Worland Strain of Beet Curly Top Virus Are Compatible with Those of the CFH Strain But Not Those of the Cal/Logan Strain

1998 ◽  
Vol 88 (11) ◽  
pp. 1174-1178 ◽  
Author(s):  
Drake C. Stenger
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Separate Species ◽  
Rep Protein ◽  
Reading Frame ◽  
Replication Assay ◽  
Beet Curly Top Virus ◽  
Rep Proteins ◽  
Cis And Trans

Cloned genomes of the CFH, Worland, and Cal/Logan strains of beet curly top virus (BCTV) served as helper viruses to trans-replicate defective (D) DNAs that are incapable of self-replication due to deletions within the C1 open reading frame encoding the replication initiator (Rep) protein. The Logan Rep protein could trans-replicate a Logan-derived D DNA in a transient replication assay conducted in Nicotiana benthamiana leaf disks. However, the Logan Rep protein was unable to trans-replicate D DNAs derived from the CFH or Worland strains. In contrast, the Rep proteins of the CFH and Worland strains could trans-replicate CFH or Worland D DNAs, but not a Logan D DNA. These results indicate that the cis- and trans-acting replication specificity elements of the CFH and Worland strains are compatible and that the three strains of BCTV may be divided into two groupings based upon replication specificity determinants. A comparison of amino acid sequences of the Rep protein for the three BCTV strains suggests that the trans-acting replication specificity element may reside in one or more of 12 amino acid residues that are identical; in two amino acid residues that are chemically similar among the CFH and Worland Rep proteins, yet are different in the Logan Rep protein; or in both. Properties including replication specificity, nucleotide sequence identity, and symptom expression were used as criteria to propose separate species designations for each of the three BCTV strains. In this proposal, the Cal/ Logan strain retains the name BCTV, CFH and the closely related Iranian isolate are designated beet severe curly top virus, and Worland is designated beet mild curly top virus.

A second expressed kininogen gene in mice

2006 ◽  
Vol 26 (2) ◽  
pp. 152-157 ◽  
Author(s):  
Edward G. Shesely ◽  
Chun-Bo Hu ◽  
François Alhenc-Gelas ◽  
Pierre Meneton ◽  
Oscar A. Carretero
Keyword(s):  
Molecular Weight ◽  
Open Reading Frame ◽  
Low Molecular Weight ◽  
High Molecular Weight Kininogen ◽  
Rt Pcr ◽  
Reading Frame ◽  
Rna Ligase ◽  
Mouse Tissues ◽  
Rapid Amplification ◽  
Race Pcr

We isolated PCR, RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE-PCR)-, and RT-PCR-generated clones from mouse kininogen family transcripts. DNA sequencing indicated that the clones were from two distinct genes. One set (K1) is from the previously reported mouse kininogen gene. The second set (K2) has an open reading frame, is 93% identical to K1 in the overlapping nucleotide sequence, and, unlike T-kininogens in the rat, encodes a bradykinin motif identical to K1. We discovered that K2 exists with two different 5′ ends. We used RT-PCR to determine the distribution and relative abundance of K1 and K2 mRNA in mouse tissues. K2 is transcribed and K1 and K2 are generally both expressed in the same tissues; however, they differ in their regulation of the alternative splicing event that yields either low-molecular-weight kininogen (LMWK) or high-molecular-weight kininogen (HMWK). For example, in the liver K1 is expressed as both HMWK and LMWK, whereas K2 is only expressed as LMWK. Conversely, in the kidney K2 is strongly expressed as both HMWK and LMWK, whereas K1 is not expressed as HMWK and expressed only very weakly as LMWK.

scholarly journals The Acinetobacter baylyi hfq Gene Encodes a Large Protein with an Unusual C Terminus

2009 ◽  
Vol 191 (17) ◽  
pp. 5553-5562 ◽  
Author(s):  
Dominik Schilling ◽  
Ulrike Gerischer
Keyword(s):  
Amino Acid ◽  
Gene Localization ◽  
Amino Acid Residues ◽  
Acinetobacter Baylyi ◽  
Reading Frame ◽  
Rich Domain ◽  
C Terminus ◽  
The Difference ◽  
Cell Phenotypes

ABSTRACT In gammaproteobacteria the Hfq protein shows a great variation in size, especially in its C-terminal part. Extremely large Hfq proteins consisting of almost 200 amino acid residues and more are found within the gammaproteobacterial family Moraxellaceae. The difference in size compared to other Hfq proteins is due to a glycine-rich domain near the C-terminal end of the protein. Acinetobacter baylyi, a nonpathogenic soil bacterium and member of the Moraxellaceae encodes a large 174-amino-acid Hfq homologue containing the unique and repetitive amino acid pattern GGGFGGQ within the glycine-rich domain. Despite the presence of the C-terminal extension, A. baylyi Hfq complemented an Escherichia coli hfq mutant in vivo. By using polyclonal anti-Hfq antibodies, we detected the large A. baylyi Hfq that corresponds to its annotated size indicating the expression and stability of the full protein. Deletion of the complete A. baylyi hfq open reading frame resulted in severe reduction of growth. In addition, a deletion or overexpression of Hfq was accompanied by the loss of cell chain assembly. The glycine-rich domain was not responsible for growth and cell phenotypes. hfq gene localization in A. baylyi is strictly conserved within the mutL-miaA-hfq operon, and we show that hfq expression starts within the preceding miaA gene or further upstream.

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