scholarly journals Cloning and analysis of a gene involved in DNA repair and recombination, the rad1 gene of Schizosaccharomyces pombe.

1990 ◽  
Vol 10 (7) ◽  
pp. 3750-3760 ◽  
Author(s):  
P Sunnerhagen ◽  
B L Seaton ◽  
A Nasim ◽  
S Subramani
Keyword(s):  
Dna Repair ◽  
Schizosaccharomyces Pombe ◽  
Protein Product ◽  
Start Codon ◽  
Insertion Mutagenesis ◽  
Amino Acid Residues ◽  
Flanking Sequence ◽  
Coding Region ◽  
Base Pairs ◽  
Repair Gene

We have cloned the rad1 gene of Schizosaccharomyces pombe by complementation of the rad1-1 mutant, which is deficient in DNA repair and recombination. The coding region of the gene is 582 base pairs long and contains no introns. The predicted product is a strongly acidic, 22-kilodalton protein containing 194 amino acid residues. This gene does not exhibit significant homology to any other known repair gene. The major transcription start site is at 27 base pairs upstream of the putative start codon. Insertion mutagenesis revealed that besides the coding region, at least 151 base pairs of 5'-flanking sequence are required for full complementing activity. A strain carrying a null allele of rad1 was constructed and found to have a phenotype closely similar to that of the rad1-1 mutant. Expression in Escherichia coli of the coding region yielded a protein product of a size close to that predicted from the DNA sequence. This product reacted with antibodies raised against a synthetic peptide with a sequence from that predicted for the protein product. We have localized the rad1 gene to NotI fragment E of the S. pombe genome.

Cloning and analysis of a gene involved in DNA repair and recombination, the rad1 gene of Schizosaccharomyces pombe

1990 ◽  
Vol 10 (7) ◽  
pp. 3750-3760
Author(s):  
P Sunnerhagen ◽  
B L Seaton ◽  
A Nasim ◽  
S Subramani
Keyword(s):  
Dna Repair ◽  
Schizosaccharomyces Pombe ◽  
Protein Product ◽  
Start Codon ◽  
Insertion Mutagenesis ◽  
Amino Acid Residues ◽  
Flanking Sequence ◽  
Coding Region ◽  
Base Pairs ◽  
Repair Gene

We have cloned the rad1 gene of Schizosaccharomyces pombe by complementation of the rad1-1 mutant, which is deficient in DNA repair and recombination. The coding region of the gene is 582 base pairs long and contains no introns. The predicted product is a strongly acidic, 22-kilodalton protein containing 194 amino acid residues. This gene does not exhibit significant homology to any other known repair gene. The major transcription start site is at 27 base pairs upstream of the putative start codon. Insertion mutagenesis revealed that besides the coding region, at least 151 base pairs of 5'-flanking sequence are required for full complementing activity. A strain carrying a null allele of rad1 was constructed and found to have a phenotype closely similar to that of the rad1-1 mutant. Expression in Escherichia coli of the coding region yielded a protein product of a size close to that predicted from the DNA sequence. This product reacted with antibodies raised against a synthetic peptide with a sequence from that predicted for the protein product. We have localized the rad1 gene to NotI fragment E of the S. pombe genome.

Identification and Characterization of the rhp23+ DNA Repair Gene in Schizosaccharomyces pombe

2000 ◽  
Vol 268 (1) ◽  
pp. 210-215 ◽  
Author(s):  
Marcel Lombaerts ◽  
Jerrelyne I. Goeloe ◽  
Hans den Dulk ◽  
Jourica A. Brandsma ◽  
Jaap Brouwer
Keyword(s):  
Dna Repair ◽  
Schizosaccharomyces Pombe ◽  
Repair Gene ◽  
Dna Repair Gene ◽  
Identification And Characterization

scholarly journals L-DOPA dioxygenase of the fly agaric toadstool: revision of the dodA gene sequence and mechanism of enzymatic pigment production

2020 ◽  
Author(s):  
Douglas M. M. Soares ◽  
Letícia C. P. Gonçalves ◽  
Caroline O. Machado ◽  
Larissa Cerrato Esteves ◽  
Cassius V. Stevani ◽  
...  
Keyword(s):  
Environmental Remediation ◽  
Kinetic Modelling ◽  
Enzymatic Catalysis ◽  
Heme Iron ◽  
Start Codon ◽  
Amino Acid Residues ◽  
Coding Region ◽  
Biotechnological Production ◽  
Pigment Formation ◽  
Alternative Start Codon

ABSTRACTl-DOPA extradiol dioxygenases (DODAs) catalyze the production of betalains and hygroaurins pigments. The sequence of the DODAs found in Caryophyllales and Basidiomycetes are not conserved, although betalains are produced both by plants and fungi. Here we revise the coding region of the dodA gene of fly agaric [Amanita muscaria (L.) Lam.] and describe an alternative start codon downstream that enables the heterologous expression of AmDODA, a promiscuous l-DOPA dioxygenase. AmDODA is 43-amino acid residues shorter than the recombinant DODA previously reported but catalyzes the formation of two isomeric seco-DOPAs that are the biosynthetic precursors of betalains and hygroaurins. The putative active site of AmDODA contains two distinct His-His-Glu motifs that can explain the dual cleavage of l-DOPA according to the mechanism proposed for non-heme iron-dependent dioxygenases. Upon addition of excess l-DOPA, both the betaxanthin and hygroaurin adducts of l-DOPA are produced. The kinetic parameters of enzymatic catalysis at pH 8.5 are similar to those reported for other l-DOPA dioxygenases. The rate constants for the conversion of l-DOPA into the betalamic acid and muscaflavin were estimated by kinetic modelling allowing the proposal of a mechanism of pigment formation. These results contribute to understanding the biosynthesis of bacterial, fungal and plant pigments, for the biotechnological production of hygroaurins, and for the development of more promiscuous dioxygenases for environmental remediation.

cDNA Catalog of Fission Yeast (Schizosaccharomyces pombe) and Its Application for Cloning of Mammalian DNA Repair Gene

1998 ◽  
pp. 115-123 ◽  
Author(s):  
Mitsuoki Morimyo ◽  
Kazuei Mita ◽  
Etsuko Hongo ◽  
Tomoyasu Higashi ◽  
Kimihiko Sugaya ◽  
...  
Keyword(s):  
Dna Repair ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Repair Gene ◽  
Dna Repair Gene

scholarly journals Optimizing Recombinant Baculovirus Vector Design for Protein Production in Insect Cells

Processes ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 2118
Author(s):  
Carina Bannach ◽  
Daniel Ruiz Buck ◽  
Genna Bobby ◽  
Leo P. Graves ◽  
Sainan Li ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Insect Cells ◽  
Recombinant Baculovirus ◽  
Gene Promoter ◽  
Protein Product ◽  
Expression Vectors ◽  
Coding Region ◽  
Base Pairs ◽  
Autographa Californica Nucleopolyhedrovirus ◽  
Transfer Vectors

Autographa californica nucleopolyhedrovirus is a very productive expression vector for recombinant proteins in insect cells. Most vectors are based on the polyhedrin gene promoter, which comprises a TAAG transcription initiation motif flanked by 20 base pairs upstream and 47 base pairs downstream before the native ATG. Many transfer vectors also include a short sequence downstream of the ATG, in which case this sequence is mutated to ATT to abolish translation. However, the ATT sequence, or AUU in the mRNA, is known to be leaky. If a target-coding region is placed in the frame with the AUU, then some products will comprise a chimeric molecule with part of the polyhedrin protein. In this study, we showed that if AUU is placed in the frame with a Strep tag and eGFP coding region, we could identify a protein product with both sequences present. Further work examined if alternative codons in lieu of AUG might reduce translation initiation further. We found that AUA was used slightly more efficiently than AUU, whereas AUC was the least efficient at initiating translation. The use of this latter codon suggested that there might also be a slight improvement of protein yield if this is incorporated into expression vectors.

scholarly journals The rad18 gene of Schizosaccharomyces pombe defines a new subgroup of the SMC superfamily involved in DNA repair.

1995 ◽  
Vol 15 (12) ◽  
pp. 7067-7080 ◽  
Author(s):  
A R Lehmann ◽  
M Walicka ◽  
D J Griffiths ◽  
J M Murray ◽  
F Z Watts ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Repair ◽  
Schizosaccharomyces Pombe ◽  
Gene Deletion ◽  
Excision Repair ◽  
Repair Pathway ◽  
Repair Gene ◽  
Dna Repair Gene ◽  
Nucleotide Excision ◽  
Biochemical Analyses

The rad18 mutant of Schizosaccharomyces pombe is very sensitive to killing by both UV and gamma radiation. We have cloned and sequenced the rad18 gene and isolated and sequenced its homolog from Saccharomyces cerevisiae, designated RHC18. The predicted Rad18 protein has all the structural properties characteristic of the SMC family of proteins, suggesting a motor function--the first implicated in DNA repair. Gene deletion shows that both rad18 and RHC18 are essential for proliferation. Genetic and biochemical analyses suggest that the product of the rad18 gene acts in a DNA repair pathway for removal of UV-induced DNA damage that is distinct from classical nucleotide excision repair. This second repair pathway involves the products of the rhp51 gene (the homolog of the RAD51 gene of S. cerevisiae) and the rad2 gene.

Nucleotide sequence of the murE gene of Escherichia coli

1989 ◽  
Vol 35 (11) ◽  
pp. 1051-1054 ◽  
Author(s):  
Jing-Song Tao ◽  
Edward E. Ishiguro
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Diaminopimelic Acid ◽  
Amino Acid Residues ◽  
Penicillin Binding Protein ◽  
Coding Region ◽  
Base Pairs ◽  
Gene Encoding ◽  
Peptidoglycan Synthesis

The nucleotide sequence of the murE gene encoding the diaminopimelic acid adding enzyme of Escherichia coli is reported. The coding region consisted of 1413 base pairs and was separated from the ftsI (penicillin-binding protein 3) gene by 61 base pairs. The deduced primary structure of MurE comprised 471 amino acid residues with a molecular mass of 50.6 kilodaltons.Key words: Escherichia coli, murE, peptidoglycan synthesis, diaminopimelic acid adding enzyme.

scholarly journals A New Recombinational DNA Repair Gene From Schizosaccharomyces pombe With Homology to Escherichia coli RecA

Genetics ◽  
1999 ◽  
Vol 152 (4) ◽  
pp. 1557-1572 ◽  
Author(s):  
Fuat K Khasanov ◽  
Galina V Savchenko ◽  
Elena V Bashkirova ◽  
Vladimir G Korolev ◽  
Wolf-Dietrich Heyer ◽  
...  
Keyword(s):  
Dna Repair ◽  
Schizosaccharomyces Pombe ◽  
Budding Yeast ◽  
Repair Gene ◽  
Acid Protein ◽  
Dna Repair Gene ◽  
Sporulation Efficiency ◽  
Highly Sensitive ◽  
Evolutionarily Conserved ◽  
Amino Acid Protein

Abstract A new DNA repair gene from Schizosaccharomyces pombe with homology to RecA was identified and characterized. Comparative analysis showed highest similarity to Saccharomyces cerevisiae Rad55p. rhp55+ (rad homologue pombe 55) encodes a predicted 350-amino-acid protein with an Mr of 38,000. The rhp55Δ mutant was highly sensitive to methyl methanesulfonate (MMS), ionizing radiation (IR), and, to a lesser degree, UV. These phenotypes were enhanced at low temperatures, similar to deletions in the S. cerevisiae RAD55 and RAD57 genes. Many rhp55Δ cells were elongated with aberrant nuclei and an increased DNA content. The rhp55 mutant showed minor deficiencies in meiotic intra- and intergenic recombination. Sporulation efficiency and spore viability were significantly reduced. Double-mutant analysis showed that rhp55+ acts in one DNA repair pathway with rhp51+ and rhp54+, homologs of the budding yeast RAD51 and RAD54 genes, respectively. However, rhp55+ is in a different epistasis group for repair of UV-, MMS-, or γ-ray-induced DNA damage than is rad22+, a putative RAD52 homolog of fission yeast. The structural and functional similarity suggests that rhp55+ is a homolog of the S. cerevisiae RAD55 gene and we propose that the functional diversification of RecA-like genes in budding yeast is evolutionarily conserved.

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